Amylases, nucleic acids encoding them and methods for making and using them

ABSTRACT

In one aspect, the invention is directed to polypeptides having an amylase activity, polynucleotides encoding the polypeptides, and methods for making and using these polynucleotides and polypeptides. In one aspect, the polypeptides of the invention can be used as amylases, for example, alpha amylases, to catalyze the hydrolysis of starch into sugars.

SEQUENCE LISTING

This application is being filed electronically via the USPTO EFS-WEBserver, as authorized and set forth in MPEP §502.05 and this electronicfiling includes an electronically submitted sequence listing; the entirecontent of this sequence listing is hereby incorporated by referenceinto the specification of this application. The sequence listing isidentified on the electronically filed ASCII (.txt) text file asfollows:

Date of File Name Creation Size SEQLISTINGD15307C2VEREN047A May 3, 201347.4 KB (48,630 bytes)

TECHNICAL FIELD

This invention relates to molecular and cellular biology andbiochemistry. In one aspect, the invention is directed to polypeptideshaving an amylase activity, e.g., alpha amylase activity,polynucleotides encoding the polypeptides, and methods for making andusing these polynucleotides and polypeptides. In one aspect, thepolypeptides of the invention can be used as amylases, for example,alpha amylases, to catalyze the hydrolysis of starch into sugars.

BACKGROUND

Starch is a complex carbohydrate often found in the human diet. Thestructure of starch is glucose polymers linked by α-1,4 and α-1,6glucosidic bonds. Amylase is an enzyme that catalyzes the hydrolysis ofstarches into sugars. Amylases hydrolyze internal β-1,4-glucosidiclinkages in starch, largely at random, to produce smaller molecularweight malto-dextrins. The breakdown of starch is important in thedigestive system and commercially. Amylases are of considerablecommercial value, being used in the initial stages (liquefaction) ofstarch processing; in wet corn milling; in alcohol production; ascleaning agents in detergent matrices; in the textile industry forstarch desizing; in baking applications; in the beverage industry; inoilfields in drilling processes; in inking of recycled paper; and inanimal feed.

Amylases are produced by a wide variety of microorganisms includingBacillus and Aspergillus, with most commercial amylases being producedfrom bacterial sources such as Bacillus licheniformis, Bacillusamyloliquefaciens, Bacillus subtilis, or Bacillus stearothermophilus. Inrecent years, the enzymes in commercial use have been those fromBacillus licheniformis because of their heat stability and performance,at least at neutral and mildly alkaline pHs.

In general, starch to fructose processing consists of four steps:liquefaction of granular starch, saccharification of the liquefiedstarch into dextrose, purification, and isomerization to fructose. Theobject of a starch liquefaction process is to convert a concentratedsuspension of starch polymer granules into a solution of soluble shorterchain length dextrins of low viscosity. This step is essential forconvenient handling with standard equipment and for efficient conversionto glucose or other sugars. To liquefy granular starch, it is necessaryto gelatinize the granules by raising the temperature of the granularstarch to over about 72° C. The heating process instantaneously disruptsthe insoluble starch granules to produce a water soluble starchsolution. The solubilized starch solution is then liquefied by amylase.A starch granule is composed of: 69-74% amylopectin, 26-31% amylose,11-14% water, 0.2-0.4% protein, 0.5-0.9% lipid, 0.05-0.1% ash,0.02-0.03% phosphorus, 0.1% pentosan. Approximately 70% of a granule isamorphous and 30% is crystalline.

A common enzymatic liquefaction process involves adjusting the pH of agranular starch slurry to between 6.0 and 6.5, the pH optimum ofalpha-amylase derived from Bacillus licheniformis, with the addition ofcalcium hydroxide, sodium hydroxide or sodium carbonate. The addition ofcalcium hydroxide has the advantage of also providing calcium ions whichare known to stabilize the alpha-amylase against inactivation. Uponaddition of alpha-amylase, the suspension is pumped through a steam jetto instantaneously raise the temperature to between 80° C. to 115° C.The starch is immediately gelatinized and, due to the presence ofalpha-amylase, depolymerized through random hydrolysis of a (1-4)glycosidic bonds by alpha-amylase to a fluid mass which is easilypumped.

In a second variation to the liquefaction process, alpha-amylase isadded to the starch suspension, the suspension is held at a temperatureof 80-100° C. to partially hydrolyze the starch granules, and thepartially hydrolyzed starch suspension is pumped through a jet attemperatures in excess of about 105° C. to thoroughly gelatinize anyremaining granular structure. After cooling the gelatinized starch, asecond addition of alpha-amylase can be made to further hydrolyze thestarch.

A third variation of this process is called the dry milling process. Indry milling, whole grain is ground and combined with water. The germ isoptionally removed by flotation separation or equivalent techniques. Theresulting mixture, which contains starch, fiber, protein and othercomponents of the grain, is liquefied using alpha-amylase. The generalpractice in the art is to undertake enzymatic liquefaction at a lowertemperature when using the dry milling process. Generally, lowtemperature liquefaction is believed to be less efficient than hightemperature liquefaction in converting starch to soluble dextrins.

Typically, after gelatinization the starch solution is held at anelevated temperature in the presence of alpha-amylase until a DE of10-20 is achieved, usually a period of 1-3 hours. Dextrose equivalent(DE) is the industry standard for measuring the concentration of totalreducing sugars, calculated as D-glucose on a dry weight basis.Unhydrolyzed granular starch has a DE of virtually zero, whereas the DEof D-glucose is defined as 100.

Corn wet milling is a process which produces corn oil, gluten meal,gluten feed and starch. Alkaline-amylase is used in the liquefaction ofstarch and glucoamylase is used in saccharification, producing glucose.Corn, a kernel of which consists of a outer seed coat (fiber), starch, acombination of starch and glucose and the inner germ, is subjected to afour step process, which results in the production of starch. The cornis steeped, de-germed, de-fibered, and finally the gluten is separated.In the steeping process, the solubles are taken out. The productremaining after removal of the solubles is de-germed, resulting inproduction of corn oil and production of an oil cake, which is added tothe solubles from the steeping step. The remaining product is de-fiberedand the fiber solids are added to the oil cake/solubles mixture. Thismixture of fiber solids, oil cake and solubles forms a gluten feed.After de-fibering, the remaining product is subjected to glutenseparation. This separation results in a gluten meal and starch. Thestarch is then subjected to liquefaction and saccharification to produceglucose.

Staling of baked products (such as bread) has been recognized as aproblem which becomes more serious as more time lies between the momentof preparation of the bread product and the moment of consumption. Theterm staling is used to describe changes undesirable to the consumer inthe properties of the bread product after leaving the oven, such as anincrease of the firmness of the crumb, a decrease of the elasticity ofthe crumb, and changes in the crust, which becomes tough and leathery.The firmness of the bread crumb increases further during storage up to alevel, which is considered as negative. The increase in crumb firmness,which is considered as the most important aspect of staling, isrecognized by the consumer a long time before the bread product hasotherwise become unsuitable for consumption.

There is a need in the industry for the identification and optimizationof amylases, useful for various uses, including commercial cornstarchliquefaction processes. These second generation acid amylases will offerimproved manufacturing and/or performance characteristics over theindustry standard enzymes from Bacillus licheniformis, for example.

There is also a need for the identification and optimization of amylaseshaving utility in automatic dish wash (ADW) products and laundrydetergent. In ADW products, the amylase will function at pH 10-11 and at45-60° C. in the presence of calcium chelators and oxidative conditions.For laundry, activity at pH 9-10 and 40° C. in the appropriate detergentmatrix will be required. Amylases are also useful in textile desizing,brewing processes, starch modification in the paper and pulp industryand other processes described in the art.

Amylases can be used commercially in the initial stages (liquefaction)of starch processing; in wet corn milling; in alcohol production; ascleaning agents in detergent matrices; in the textile industry forstarch desizing; in baking applications; in the beverage industry; inoilfields in drilling processes; in inking of recycled paper and inanimal feed. Amylases are also useful in textile desizing, brewingprocesses, starch modification in the paper and pulp industry and otherprocesses.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the invention is notentitled to antedate such disclosure by virtue of prior invention.

SUMMARY

The invention provides isolated or recombinant nucleic acids comprisinga nucleic acid sequence having at least about 50%, 51%, 52%, 53%, 54%,55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%,69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or more, or complete (100%) sequence identity to anexemplary nucleic acid of the invention over a region of at least about50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700,750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350,1400, 1450, 1500, 1550 or more, residues, wherein the nucleic acidencodes at least one polypeptide having an amylase activity, and thesequence identities are determined by analysis with a sequencecomparison algorithm or by a visual inspection. Exemplary nucleic acidsof the invention include isolated or recombinant nucleic acidscomprising a nucleic acid sequence as set forth in SEQ ID NO:1, andsubsequences thereof, e.g., at least about 10, 15, 20, 25, 30, 35, 40,45, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650,700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300,1350, 1400, 1450, 1500 or more residues in length, or over the fulllength of a gene or transcript.

Exemplary nucleic acids of the invention also include isolated orrecombinant nucleic acids encoding a polypeptide having a sequence asset forth in SEQ ID NO:2, and subsequences thereof and variants thereof.In one aspect, the polypeptide has an amylase activity, e.g., an alphaamylase activity.

In one aspect, the invention also provides amylase-encoding nucleicacids with a common novelty in that they are derived from mixedcultures. The invention provides amylase-encoding nucleic acids isolatedfrom mixed cultures comprising a nucleic acid sequence having at leastabout 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%,63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%)sequence identity to an exemplary nucleic acid of the invention over aregion of at least about 50, 75, 100, 150, 200, 250, 300, 350, 400, 450,500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100,1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550 or more, residues,wherein the nucleic acid encodes at least one polypeptide having anamylase activity, and the sequence identities are determined by analysiswith a sequence comparison algorithm or by a visual inspection. In oneaspect, the invention provides amylase-encoding nucleic acids isolatedfrom mixed cultures comprising a nucleic acid sequence as set forth inSEQ ID NO:1, and subsequences thereof, e.g., at least about 10, 15, 20,25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500,550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150,1200, 1250, 1300, 1350, 1400, 1450, 1500 or more residues in length, orover the full length of a gene or transcript.

In one aspect, the invention also provides amylase-encoding nucleicacids with a common novelty in that they are derived from environmentalsources, e.g., mixed environmental sources. In one aspect, the inventionprovides amylase-encoding nucleic acids isolated from environmentalsources, e.g., mixed environmental sources, comprising a nucleic acidsequence having at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ormore, or complete (100%) sequence identity to an exemplary nucleic acidof the invention over a region of at least about 50, 75, 100, 150, 200,250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900,950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500,1550 or more, residues, wherein the nucleic acid encodes at least onepolypeptide having an amylase activity, and the sequence identities aredetermined by analysis with a sequence comparison algorithm or by avisual inspection. In one aspect, the invention providesamylase-encoding nucleic acids isolated from environmental sources,e.g., mixed environmental sources, comprising a nucleic acid sequence asset forth in SEQ ID NO:1, and subsequences thereof, e.g., at least about10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250, 300, 350,400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050,1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500 or more residues inlength, or over the full length of a gene or transcript.

In one aspect, the sequence comparison algorithm is a BLAST version2.2.2 algorithm where a filtering setting is set to blastall-p blastp-d“nr pataa”-F F, and all other options are set to default.

Another aspect of the invention is an isolated or recombinant nucleicacid including at least 10 consecutive bases of a nucleic acid sequenceof the invention, sequences substantially identical thereto, and thesequences complementary thereto.

In one aspect, the amylase activity comprises α-amylase activity,including the ability to hydrolyze internal alpha-1,4-glucosidiclinkages in starch to produce smaller molecular weight malto-dextrins.In one aspect, the α-amylase activity includes hydrolyzing internalalpha-1,4-glucosidic linkages in starch at random. The amylase activitycan comprise a glucoamylase activity, a 1,4-α-D-glucan glucohydrolaseactivity, an α-amylase activity, an exoamylase activity, or β-amylaseactivity.

The amylase activity can comprise hydrolyzing glucosidic bonds. In oneaspect, the glucosidic bonds comprise an α-1,4-glucosidic bond. Inanother aspect, the glucosidic bonds comprise an α-1,6-glucosidic bond.In one aspect, the amylase activity comprises hydrolyzing glucosidicbonds in starch, e.g., liquefied starch. The amylase activity canfurther comprise hydrolyzing glucosidic bonds into maltodextrins. In oneaspect, the amylase activity comprises cleaving a maltose or a D-glucoseunit from non-reducing end of the starch.

In one aspect, the isolated or recombinant nucleic acid encodes apolypeptide having an amylase activity which is thermostable. Thepolypeptide can retain an amylase activity under conditions comprising atemperature range of between about 37° C. to about 95° C.; between about55° C. to about 85° C., between about 70° C. to about 95° C., or,between about 90° C. to about 95° C.

In another aspect, the isolated or recombinant nucleic acid encodes apolypeptide having an amylase activity which is thermotolerant. Thepolypeptide can retain an amylase activity after exposure to atemperature in the range from greater than 37° C. to about 95° C. oranywhere in the range from greater than 55° C. to about 85° C. In oneaspect, the polypeptide retains an amylase activity after exposure to atemperature in the range from greater than 90° C. to about 95° C. at pH4.5.

The invention provides isolated or recombinant nucleic acids comprisinga sequence that hybridizes under stringent conditions to a nucleic acidcomprising a sequence as set forth in SEQ ID NO:1, or fragments orsubsequences thereof. In one aspect, the nucleic acid encodes apolypeptide having an amylase activity. The nucleic acid can be at leastabout 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600,650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250,1300, 1350, 1400, 1450, 1500 or more residues in length or the fulllength of the gene or transcript. In one aspect, the stringentconditions include a wash step comprising a wash in 0.2×SSC at atemperature of about 65° C. for about 15 minutes.

The invention provides a nucleic acid probe for identifying a nucleicacid encoding a polypeptide having an amylase activity, wherein theprobe comprises at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55,60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450,500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 or more,consecutive bases of a sequence comprising a sequence of the invention,or fragments or subsequences thereof, wherein the probe identifies thenucleic acid by binding or hybridization. The probe can comprise anoligonucleotide comprising at least about 10 to 50, about 20 to 60,about 30 to 70, about 40 to 80, or about 60 to 100 consecutive bases ofa sequence comprising a sequence of the invention, or fragments orsubsequences thereof.

The invention provides a nucleic acid probe for identifying a nucleicacid encoding a polypeptide having an amylase activity, wherein theprobe comprises a nucleic acid comprising a sequence at least about 10,15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400,450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 or moreresidues having at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ormore, or complete (100%) sequence identity to a nucleic acid of theinvention, wherein the sequence identities are determined by analysiswith a sequence comparison algorithm or by visual inspection.

The probe can comprise an oligonucleotide comprising at least about 10to 50, about 20 to 60, about 30 to 70, about 40 to 80, or about 60 to100 consecutive bases of a nucleic acid sequence of the invention, or asubsequence thereof.

The invention provides an amplification primer sequence pair foramplifying a nucleic acid encoding a polypeptide having an amylaseactivity, wherein the primer pair is capable of amplifying a nucleicacid comprising a sequence of the invention, or fragments orsubsequences thereof. One or each member of the amplification primersequence pair can comprise an oligonucleotide comprising at least about10 to 50 consecutive bases of the sequence.

The invention provides methods of amplifying a nucleic acid encoding apolypeptide having an amylase activity comprising amplification of atemplate nucleic acid with an amplification primer sequence pair capableof amplifying a nucleic acid sequence of the invention, or fragments orsubsequences thereof.

The invention provides expression cassettes comprising a nucleic acid ofthe invention or a subsequence thereof. In one aspect, the expressioncassette can comprise the nucleic acid that is operably linked to apromoter. The promoter can be a viral, bacterial, mammalian or plantpromoter. In one aspect, the plant promoter can be a potato, rice, corn,wheat, tobacco or barley promoter. The promoter can be a constitutivepromoter. The constitutive promoter can comprise CaMV35S. In anotheraspect, the promoter can be an inducible promoter. In one aspect, thepromoter can be a tissue-specific promoter or an environmentallyregulated or a developmentally regulated promoter. Thus, the promotercan be, e.g., a seed-specific, a leaf-specific, a root-specific, astem-specific or an abscission-induced promoter. In one aspect, theexpression cassette can further comprise a plant or plant virusexpression vector.

The invention provides cloning vehicles comprising an expressioncassette (e.g., a vector) of the invention or a nucleic acid of theinvention. The cloning vehicle can be a viral vector, a plasmid, aphage, a phagemid, a cosmid, a fosmid, a bacteriophage or an artificialchromosome. The viral vector can comprise an adenovirus vector, aretroviral vector or an adeno-associated viral vector. The cloningvehicle can comprise a bacterial artificial chromosome (BAC), a plasmid,a bacteriophage P1-derived vector (PAC), a yeast artificial chromosome(YAC), or a mammalian artificial chromosome (MAC).

The invention provides transformed cell comprising a nucleic acid of theinvention or an expression cassette (e.g., a vector) of the invention,or a cloning vehicle of the invention. In one aspect, the transformedcell can be a bacterial cell, a mammalian cell, a fungal cell, a yeastcell, an insect cell or a plant cell. In one aspect, the plant cell canbe a potato, wheat, rice, corn, tobacco or barley cell.

The invention provides transgenic non-human animals comprising a nucleicacid of the invention or an expression cassette (e.g., a vector) of theinvention. In one aspect, the animal is a mouse.

The invention provides transgenic plants comprising a nucleic acid ofthe invention or an expression cassette (e.g., a vector) of theinvention. The transgenic plant can be a corn plant, a potato plant, atomato plant, a wheat plant, an oilseed plant, a rapeseed plant, asoybean plant, a rice plant, a barley plant or a tobacco plant.

The invention provides transgenic seeds comprising a nucleic acid of theinvention or an expression cassette (e.g., a vector) of the invention.The transgenic seed can be a corn seed, a wheat kernel, an oilseed, arapeseed, a soybean seed, a palm kernel, a sunflower seed, a sesameseed, a peanut or a tobacco plant seed.

The invention provides an antisense oligonucleotide comprising a nucleicacid sequence complementary to or capable of hybridizing under stringentconditions to a nucleic acid of the invention. The invention providesmethods of inhibiting the translation of an amylase message in a cellcomprising administering to the cell or expressing in the cell anantisense oligonucleotide comprising a nucleic acid sequencecomplementary to or capable of hybridizing under stringent conditions toa nucleic acid of the invention.

The invention provides an isolated or recombinant polypeptide comprisingan amino acid sequence having at least about 50%, 51%, 52%, 53%, 54%,55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%,69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or more, or complete (100%) sequence identity to anexemplary polypeptide or peptide of the invention over a region of atleast about 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550,600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200,1250, 1300, 1350, 1400, 1450, 1500, 1550 or more residues, or over thefull length of the polypeptide, and the sequence identities aredetermined by analysis with a sequence comparison algorithm or by avisual inspection. Exemplary polypeptide or peptide sequences of theinvention include SEQ ID NO:2, and subsequences thereof and variantsthereof, e.g., at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 75,100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750,800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350,1400, 1450, 1500 or more residues in length, or over the full length ofan enzyme. Exemplary polypeptide or peptide sequences of the inventioninclude sequence encoded by a nucleic acid of the invention. Exemplarypolypeptide or peptide sequences of the invention include polypeptidesor peptides specifically bound by an antibody of the invention. In oneaspect, a polypeptide of the invention has at least one amylaseactivity, e.g., an alpha amylase activity.

Another aspect of the invention is an isolated or recombinantpolypeptide or peptide including at least 10 consecutive bases of apolypeptide or peptide sequence of the invention, sequencessubstantially identical thereto, and the sequences complementarythereto.

In one aspect, the amylase activity of a polypeptide or peptide of theinvention comprises an α-amylase activity, including the ability tohydrolyze internal alpha-1,4-glucosidic linkages in starch to producesmaller molecular weight malto-dextrins. In one aspect, the α-amylaseactivity includes hydrolyzing internal alpha-1,4-glucosidic linkages instarch at random. The amylase activity can comprise a glucoamylaseactivity, a 1,4-α-D-glucan glucohydrolase activity, an α-amylaseactivity, an exoamylase activity, or β-amylase activity. The amylaseactivity can comprise hydrolyzing glucosidic bonds. In one aspect, theglucosidic bonds comprise an α-1,4-glucosidic bond. In another aspect,the glucosidic bonds comprise an α-1,6-glucosidic bond. In one aspect,the amylase activity comprises hydrolyzing glucosidic bonds in starch,e.g., liquefied starch. The amylase activity can further comprisehydrolyzing glucosidic bonds into maltodextrins. In one aspect, theamylase activity comprises cleaving a maltose or a D-glucose unit fromnon-reducing end of the starch.

In one aspect, the amylase activity can be thermostable. The polypeptidecan retain an amylase activity under conditions comprising a temperaturerange of between about 37° C. to about 95° C., between about 55° C. toabout 85° C., between about 70° C. to about 95° C., or between about 90°C. to about 95° C. In another aspect, the amylase activity can bethermotolerant. The polypeptide can retain an amylase activity afterexposure to a temperature in the range from greater than 37° C. to about95° C., or in the range from greater than 55° C. to about 85° C. In oneaspect, the polypeptide can retain an amylase activity after exposure toa temperature in the range from greater than 90° C. to about 95° C. atpH 4.5.

In one aspect, the isolated or recombinant polypeptide can comprise thepolypeptide of the invention that lacks a signal sequence. In oneaspect, the isolated or recombinant polypeptide can comprise thepolypeptide of the invention comprising a heterologous signal sequence,such as a heterologous amylase or non-amylase signal sequence.

In one aspect, the invention provides a signal sequence comprising anamino terminal peptide of SEQ ID NO:2. In one aspect, the inventionprovides a signal sequence consisting of a peptide having a subsequenceof SEQ ID NO:2. In one aspect, the invention provides chimeric proteinscomprising a first domain comprising a signal sequence of the inventionand at least a second domain. The protein can be a fusion protein. Thesecond domain can comprise an enzyme. The enzyme can be an amylase(e.g., an amylase of the invention, or, another amylase).

In one aspect, the amylase activity comprises a specific activity atabout 37° C. in the range from about 100 to about 1000 units permilligram of protein. In another aspect, the amylase activity comprisesa specific activity from about 500 to about 750 units per milligram ofprotein. Alternatively, the amylase activity comprises a specificactivity at 37° C. in the range from about 500 to about 1200 units permilligram of protein. In one aspect, the amylase activity comprises aspecific activity at 37° C. in the range from about 750 to about 1000units per milligram of protein. In another aspect, the thermotolerancecomprises retention of at least half of the specific activity of theamylase at 37° C. after being heated to the elevated temperature.Alternatively, the thermotolerance can comprise retention of specificactivity at 37° C. in the range from about 500 to about 1200 units permilligram of protein after being heated to the elevated temperature.

The invention provides the isolated or recombinant polypeptide of theinvention, wherein the polypeptide comprises at least one glycosylationsite. In one aspect, glycosylation can be an N-linked glycosylation. Inone aspect, the polypeptide can be glycosylated after being expressed ina P. pastoris or a S. pombe.

In one aspect, the polypeptide can retain an amylase activity underconditions comprising about pH 6.5, pH 6, pH 5.5, pH 5, pH 4.5 or pH 4.In another aspect, the polypeptide can retain an amylase activity underconditions comprising about pH 7, pH 7.5 pH 8.0, pH 8.5, pH 9, pH 9.5,pH 10, pH 10.5 or pH 11.

The invention provides protein preparations comprising a polypeptide ofthe invention, wherein the protein preparation comprises a liquid, asolid or a gel.

The invention provides heterodimers comprising a polypeptide of theinvention and a second domain. In one aspect, the second domain can be apolypeptide and the heterodimer can be a fusion protein. In one aspect,the second domain can be an epitope or a tag. In one aspect, theinvention provides homodimers comprising a polypeptide of the invention.

The invention provides immobilized polypeptides having an amylaseactivity, wherein the polypeptide comprises a polypeptide of theinvention, a polypeptide encoded by a nucleic acid of the invention, ora polypeptide comprising a polypeptide of the invention and a seconddomain. In one aspect, the polypeptide can be immobilized on a cell, ametal, a resin, a polymer, a ceramic, a glass, a microelectrode, agraphitic particle, a bead, a gel, a plate, an array or a capillarytube.

The invention provides arrays comprising an immobilized nucleic acid ofthe invention. The invention provides arrays comprising an antibody ofthe invention.

The invention provides isolated or recombinant antibodies thatspecifically bind to a polypeptide of the invention or to a polypeptideencoded by a nucleic acid of the invention. The antibody can be amonoclonal or a polyclonal antibody. The invention provides hybridomascomprising an antibody of the invention, e.g., an antibody thatspecifically binds to a polypeptide of the invention or to a polypeptideencoded by a nucleic acid of the invention.

The invention provides food supplements for an animal comprising apolypeptide of the invention, e.g., a polypeptide encoded by the nucleicacid of the invention. In one aspect, the polypeptide in the foodsupplement can be glycosylated. The invention provides edible enzymedelivery matrices comprising a polypeptide of the invention, e.g., apolypeptide encoded by the nucleic acid of the invention. In one aspect,the delivery matrix comprises a pellet. In one aspect, the polypeptidecan be glycosylated. In one aspect, the amylase activity isthermotolerant. In another aspect, the amylase activity is thermostable.

The invention provides method of isolating or identifying a polypeptidehaving an amylase activity comprising the steps of: (a) providing anantibody of the invention; (b) providing a sample comprisingpolypeptides; and (c) contacting the sample of step (b) with theantibody of step (a) under conditions wherein the antibody canspecifically bind to the polypeptide, thereby isolating or identifying apolypeptide having an amylase activity.

The invention provides methods of making an anti-amylase antibodycomprising administering to a non-human animal a nucleic acid of theinvention or a polypeptide of the invention or subsequences thereof inan amount sufficient to generate a humoral immune response, therebymaking an anti-amylase antibody. The invention provides methods ofmaking an anti-amylase immune comprising administering to a non-humananimal a nucleic acid of the invention or a polypeptide of the inventionor subsequences thereof in an amount sufficient to generate an immuneresponse.

The invention provides methods of producing a recombinant polypeptidecomprising the steps of: (a) providing a nucleic acid of the inventionoperably linked to a promoter; and (b) expressing the nucleic acid ofstep (a) under conditions that allow expression of the polypeptide,thereby producing a recombinant polypeptide. In one aspect, the methodcan further comprise transforming a host cell with the nucleic acid ofstep (a) followed by expressing the nucleic acid of step (a), therebyproducing a recombinant polypeptide in a transformed cell.

The invention provides methods for identifying a polypeptide having anamylase activity comprising the following steps: (a) providing apolypeptide of the invention; or a polypeptide encoded by a nucleic acidof the invention; (b) providing an amylase substrate; and (c) contactingthe polypeptide or a fragment or variant thereof of step (a) with thesubstrate of step (b) and detecting a decrease in the amount ofsubstrate or an increase in the amount of a reaction product, wherein adecrease in the amount of the substrate or an increase in the amount ofthe reaction product detects a polypeptide having an amylase activity.In one aspect, the substrate can be a starch, e.g., a liquefied starch.

The invention provides methods for identifying an amylase substratecomprising the following steps: (a) providing a polypeptide of theinvention; or a polypeptide encoded by a nucleic acid of the invention;(b) providing a test substrate; and (c) contacting the polypeptide ofstep (a) with the test substrate of step (b) and detecting a decrease inthe amount of substrate or an increase in the amount of reactionproduct, wherein a decrease in the amount of the substrate or anincrease in the amount of a reaction product identifies the testsubstrate as an amylase substrate.

The invention provides methods of determining whether a test compoundspecifically binds to a polypeptide comprising the following steps: (a)expressing a nucleic acid or a vector comprising the nucleic acid underconditions permissive for translation of the nucleic acid to apolypeptide, wherein the nucleic acid comprises a nucleic acid of theinvention, or, providing a polypeptide of the invention; (b) providing atest compound; (c) contacting the polypeptide with the test compound;and (d) determining whether the test compound of step (b) specificallybinds to the polypeptide.

The invention provides methods for identifying a modulator of an amylaseactivity comprising the following steps: (a) providing a polypeptide ofthe invention or a polypeptide encoded by a nucleic acid of theinvention; (b) providing a test compound; (c) contacting the polypeptideof step (a) with the test compound of step (b) and measuring an activityof the amylase, wherein a change in the amylase activity measured in thepresence of the test compound compared to the activity in the absence ofthe test compound provides a determination that the test compoundmodulates the amylase activity. In one aspect, the amylase activity canbe measured by providing an amylase substrate and detecting a decreasein the amount of the substrate or an increase in the amount of areaction product, or, an increase in the amount of the substrate or adecrease in the amount of a reaction product. A decrease in the amountof the substrate or an increase in the amount of the reaction productwith the test compound as compared to the amount of substrate orreaction product without the test compound identifies the test compoundas an activator of amylase activity. An increase in the amount of thesubstrate or a decrease in the amount of the reaction product with thetest compound as compared to the amount of substrate or reaction productwithout the test compound identifies the test compound as an inhibitorof amylase activity.

The invention provides computer systems comprising a processor and adata storage device wherein said data storage device has stored thereona polypeptide sequence or a nucleic acid sequence of the invention(e.g., a polypeptide encoded by a nucleic acid of the invention). In oneaspect, the computer system can further comprise a sequence comparisonalgorithm and a data storage device having at least one referencesequence stored thereon. In another aspect, the sequence comparisonalgorithm comprises a computer program that indicates polymorphisms. Inone aspect, the computer system can further comprise an identifier thatidentifies one or more features in said sequence. The invention providescomputer readable media having stored thereon a polypeptide sequence ora nucleic acid sequence of the invention. The invention provides methodsfor identifying a feature in a sequence comprising the steps of: (a)reading the sequence using a computer program which identifies one ormore features in a sequence, wherein the sequence comprises apolypeptide sequence or a nucleic acid sequence of the invention; and(b) identifying one or more features in the sequence with the computerprogram. The invention provides methods for comparing a first sequenceto a second sequence comprising the steps of: (a) reading the firstsequence and the second sequence through use of a computer program whichcompares sequences, wherein the first sequence comprises a polypeptidesequence or a nucleic acid sequence of the invention; and (b)determining differences between the first sequence and the secondsequence with the computer program. The step of determining differencesbetween the first sequence and the second sequence can further comprisethe step of identifying polymorphisms. In one aspect, the method canfurther comprise an identifier that identifies one or more features in asequence. In another aspect, the method can comprise reading the firstsequence using a computer program and identifying one or more featuresin the sequence.

The invention provides methods for isolating or recovering a nucleicacid encoding a polypeptide having an amylase activity from anenvironmental sample comprising the steps of: (a) providing anamplification primer sequence pair for amplifying a nucleic acidencoding a polypeptide having an amylase activity, wherein the primerpair is capable of amplifying a nucleic acid of the invention; (b)isolating a nucleic acid from the environmental sample or treating theenvironmental sample such that nucleic acid in the sample is accessiblefor hybridization to the amplification primer pair; and, (c) combiningthe nucleic acid of step (b) with the amplification primer pair of step(a) and amplifying nucleic acid from the environmental sample, therebyisolating or recovering a nucleic acid encoding a polypeptide having anamylase activity from an environmental sample. One or each member of theamplification primer sequence pair can comprise an oligonucleotidecomprising at least about 10 to 50 consecutive bases of a sequence ofthe invention.

The invention provides methods for isolating or recovering a nucleicacid encoding a polypeptide having an amylase activity from anenvironmental sample comprising the steps of: (a) providing apolynucleotide probe comprising a nucleic acid of the invention or asubsequence thereof; (b) isolating a nucleic acid from the environmentalsample or treating the environmental sample such that nucleic acid inthe sample is accessible for hybridization to a polynucleotide probe ofstep (a); (c) combining the isolated nucleic acid or the treatedenvironmental sample of step (b) with the polynucleotide probe of step(a); and (d) isolating a nucleic acid that specifically hybridizes withthe polynucleotide probe of step (a), thereby isolating or recovering anucleic acid encoding a polypeptide having an amylase activity from anenvironmental sample. The environmental sample can comprise a watersample, a liquid sample, a soil sample, an air sample or a biologicalsample. In one aspect, the biological sample can be derived from abacterial cell, a protozoan cell, an insect cell, a yeast cell, a plantcell, a fungal cell or a mammalian cell.

The invention provides methods of generating a variant of a nucleic acidencoding a polypeptide having an amylase activity comprising the stepsof: (a) providing a template nucleic acid comprising a nucleic acid ofthe invention; and (b) modifying, deleting or adding one or morenucleotides in the template sequence, or a combination thereof, togenerate a variant of the template nucleic acid. In one aspect, themethod can further comprise expressing the variant nucleic acid togenerate a variant amylase polypeptide. The modifications, additions ordeletions can be introduced by a method comprising error-prone PCR,shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexualPCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursiveensemble mutagenesis, exponential ensemble mutagenesis, site-specificmutagenesis, gene reassembly, gene site saturated mutagenesis (GSSM),synthetic ligation reassembly (SLR) or a combination thereof. In anotheraspect, the modifications, additions or deletions are introduced by amethod comprising recombination, recursive sequence recombination,phosphothioate-modified DNA mutagenesis, uracil-containing templatemutagenesis, gapped duplex mutagenesis, point mismatch repairmutagenesis, repair-deficient host strain mutagenesis, chemicalmutagenesis, radiogenic mutagenesis, deletion mutagenesis,restriction-selection mutagenesis, restriction-purification mutagenesis,artificial gene synthesis, ensemble mutagenesis, chimeric nucleic acidmultimer creation and a combination thereof.

In one aspect, the method can be iteratively repeated until an amylasehaving an altered or different activity or an altered or differentstability from that of a polypeptide encoded by the template nucleicacid is produced. In one aspect, the variant amylase polypeptide isthermotolerant, and retains some activity after being exposed to anelevated temperature. In another aspect, the variant amylase polypeptidehas increased glycosylation as compared to the amylase encoded by atemplate nucleic acid. Alternatively, the variant amylase polypeptidehas an amylase activity under a high temperature, wherein the amylaseencoded by the template nucleic acid is not active under the hightemperature. In one aspect, the method can be iteratively repeated untilan amylase coding sequence having an altered codon usage from that ofthe template nucleic acid is produced. In another aspect, the method canbe iteratively repeated until an amylase gene having higher or lowerlevel of message expression or stability from that of the templatenucleic acid is produced.

The invention provides methods for modifying codons in a nucleic acidencoding a polypeptide having an amylase activity to increase itsexpression in a host cell, the method comprising the following steps:(a) providing a nucleic acid of the invention encoding a polypeptidehaving an amylase activity; and, (b) identifying a non-preferred or aless preferred codon in the nucleic acid of step (a) and replacing itwith a preferred or neutrally used codon encoding the same amino acid asthe replaced codon, wherein a preferred codon is a codonover-represented in coding sequences in genes in the host cell and anon-preferred or less preferred codon is a codon under-represented incoding sequences in genes in the host cell, thereby modifying thenucleic acid to increase its expression in a host cell.

The invention provides methods for modifying codons in a nucleic acidencoding a polypeptide having an amylase activity; the method comprisingthe following steps: (a) providing a nucleic acid of the invention; and,(b) identifying a codon in the nucleic acid of step (a) and replacing itwith a different codon encoding the same amino acid as the replacedcodon, thereby modifying codons in a nucleic acid encoding an amylase.

The invention provides methods for modifying codons in a nucleic acidencoding a polypeptide having an amylase activity to increase itsexpression in a host cell, the method comprising the following steps:(a) providing a nucleic acid of the invention encoding an amylasepolypeptide; and, (b) identifying a non-preferred or a less preferredcodon in the nucleic acid of step (a) and replacing it with a preferredor neutrally used codon encoding the same amino acid as the replacedcodon, wherein a preferred codon is a codon over-represented in codingsequences in genes in the host cell and a non-preferred or lesspreferred codon is a codon under-represented in coding sequences ingenes in the host cell, thereby modifying the nucleic acid to increaseits expression in a host cell.

The invention provides methods for modifying a codon in a nucleic acidencoding a polypeptide having an amylase activity to decrease itsexpression in a host cell, the method comprising the following steps:(a) providing a nucleic acid of the invention; and (b) identifying atleast one preferred codon in the nucleic acid of step (a) and replacingit with a non-preferred or less preferred codon encoding the same aminoacid as the replaced codon, wherein a preferred codon is a codonover-represented in coding sequences in genes in a host cell and anon-preferred or less preferred codon is a codon under-represented incoding sequences in genes in the host cell, thereby modifying thenucleic acid to decrease its expression in a host cell. In one aspect,the host cell can be a bacterial cell, a fungal cell, an insect cell, ayeast cell, a plant cell or a mammalian cell.

The invention provides methods for producing a library of nucleic acidsencoding a plurality of modified amylase active sites or substratebinding sites, wherein the modified active sites or substrate bindingsites are derived from a first nucleic acid comprising a sequenceencoding a first active site or a first substrate binding site themethod comprising the following steps: (a) providing a first nucleicacid encoding a first active site or first substrate binding site,wherein the first nucleic acid sequence comprises a sequence thathybridizes under stringent conditions to a nucleic acid of theinvention, and the nucleic acid encodes an amylase active site or anamylase substrate binding site; (b) providing a set of mutagenicoligonucleotides that encode naturally-occurring amino acid variants ata plurality of targeted codons in the first nucleic acid; and, (c) usingthe set of mutagenic oligonucleotides to generate a set of activesite-encoding or substrate binding site-encoding variant nucleic acidsencoding a range of amino acid variations at each amino acid codon thatwas mutagenized, thereby producing a library of nucleic acids encoding aplurality of modified amylase active sites or substrate binding sites.In one aspect, the method comprises mutagenizing the first nucleic acidof step (a) by a method comprising an optimized directed evolutionsystem, gene site-saturation mutagenesis (GSSM), synthetic ligationreassembly (SLR), error-prone PCR, shuffling, oligonucleotide-directedmutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis,cassette mutagenesis, recursive ensemble mutagenesis, exponentialensemble mutagenesis, site-specific mutagenesis, gene reassembly, genesite saturated mutagenesis (GSSM), synthetic ligation reassembly (SLR)and a combination thereof. In another aspect, the method comprisesmutagenizing the first nucleic acid of step (a) or variants by a methodcomprising recombination, recursive sequence recombination,phosphothioate-modified DNA mutagenesis, uracil-containing templatemutagenesis, gapped duplex mutagenesis, point mismatch repairmutagenesis, repair-deficient host strain mutagenesis, chemicalmutagenesis, radiogenic mutagenesis, deletion mutagenesis,restriction-selection mutagenesis, restriction-purification mutagenesis,artificial gene synthesis, ensemble mutagenesis, chimeric nucleic acidmultimer creation and a combination thereof.

The invention provides methods for making a small molecule comprisingthe following steps: (a) providing a plurality of biosynthetic enzymescapable of synthesizing or modifying a small molecule, wherein one ofthe enzymes comprises an amylase enzyme encoded by a nucleic acid of theinvention; (b) providing a substrate for at least one of the enzymes ofstep (a); and (c) reacting the substrate of step (b) with the enzymesunder conditions that facilitate a plurality of biocatalytic reactionsto generate a small molecule by a series of biocatalytic reactions. Theinvention provides methods for modifying a small molecule comprising thefollowing steps: (a) providing an amylase enzyme, wherein the enzymecomprises a polypeptide of the invention, or, a polypeptide encoded by anucleic acid of the invention, or a subsequence thereof; (b) providing asmall molecule; and (c) reacting the enzyme of step (a) with the smallmolecule of step (b) under conditions that facilitate an enzymaticreaction catalyzed by the amylase enzyme, thereby modifying a smallmolecule by an amylase enzymatic reaction. In one aspect, the method cancomprise a plurality of small molecule substrates for the enzyme of step(a), thereby generating a library of modified small molecules producedby at least one enzymatic reaction catalyzed by the amylase enzyme. Inone aspect, the method can comprise a plurality of additional enzymesunder conditions that facilitate a plurality of biocatalytic reactionsby the enzymes to form a library of modified small molecules produced bythe plurality of enzymatic reactions. In another aspect, the method canfurther comprise the step of testing the library to determine if aparticular modified small molecule which exhibits a desired activity ispresent within the library. The step of testing the library can furthercomprise the steps of systematically eliminating all but one of thebiocatalytic reactions used to produce a portion of the plurality of themodified small molecules within the library by testing the portion ofthe modified small molecule for the presence or absence of theparticular modified small molecule with a desired activity, andidentifying at least one specific biocatalytic reaction that producesthe particular modified small molecule of desired activity.

The invention provides methods for determining a functional fragment ofan amylase enzyme comprising the steps of: (a) providing an amylaseenzyme, wherein the enzyme comprises a polypeptide of the invention, ora polypeptide encoded by a nucleic acid of the invention, or asubsequence thereof; and (b) deleting a plurality of amino acid residuesfrom the sequence of step (a) and testing the remaining subsequence foran amylase activity, thereby determining a functional fragment of anamylase enzyme. In one aspect, the amylase activity is measured byproviding an amylase substrate and detecting a decrease in the amount ofthe substrate or an increase in the amount of a reaction product.

The invention provides methods for whole cell engineering of new ormodified phenotypes by using real-time metabolic flux analysis, themethod comprising the following steps: (a) making a modified cell bymodifying the genetic composition of a cell, wherein the geneticcomposition is modified by addition to the cell of a nucleic acid of theinvention; (b) culturing the modified cell to generate a plurality ofmodified cells; (c) measuring at least one metabolic parameter of thecell by monitoring the cell culture of step (b) in real time; and, (d)analyzing the data of step (c) to determine if the measured parameterdiffers from a comparable measurement in an unmodified cell undersimilar conditions, thereby identifying an engineered phenotype in thecell using real-time metabolic flux analysis. In one aspect, the geneticcomposition of the cell can be modified by a method comprising deletionof a sequence or modification of a sequence in the cell, or, knockingout the expression of a gene. In one aspect, the method can furthercomprise selecting a cell comprising a newly engineered phenotype. Inanother aspect, the method can comprise culturing the selected cell,thereby generating a new cell strain comprising a newly engineeredphenotype.

The invention provides methods for hydrolyzing a starch comprising thefollowing steps: (a) providing a polypeptide having an amylase activity,wherein the polypeptide comprises a polypeptide of the invention; (b)providing a composition comprising a starch; and (c) contacting thepolypeptide of step (a) with the composition of step (b) underconditions wherein the polypeptide hydrolyzes the starch. In one aspect,the composition comprising starch that comprises an α-1,4-glucosidicbond or an α-1,6-glucosidic bond. In one aspect, the amylase activity isan α-amylase activity. In one aspect, the α-amylase activity hydrolyzesinternal bonds in a starch or other polysaccharide.

The invention provides methods for liquefying or removing a starch froma composition comprising the following steps: (a) providing apolypeptide having an amylase activity, wherein the polypeptidecomprises a polypeptide of the invention; (b) providing a compositioncomprising a starch; and (c) contacting the polypeptide of step (a) withthe composition of step (b) under conditions wherein the polypeptideremoves or liquefies the starch.

The invention provides methods of increasing thermotolerance orthermostability of an amylase polypeptide, the method comprisingglycosylating an amylase polypeptide, wherein the polypeptide comprisesat least thirty contiguous amino acids of a polypeptide of theinvention; or a polypeptide encoded by a nucleic acid sequence of theinvention, thereby increasing the thermotolerance or thermostability ofthe amylase polypeptide. In one aspect, the amylase specific activitycan be thermostable or thermotolerant at a temperature in the range fromgreater than about 37° C. to about 95° C.

The invention provides methods for overexpressing a recombinant amylasepolypeptide in a cell comprising expressing a vector comprising anucleic acid comprising a nucleic acid of the invention or a nucleicacid sequence of the invention, wherein the sequence identities aredetermined by analysis with a sequence comparison algorithm or by visualinspection, wherein overexpression is effected by use of a high activitypromoter, a dicistronic vector or by gene amplification of the vector.

The invention provides detergent compositions comprising a polypeptideof the invention or a polypeptide encoded by a nucleic acid of theinvention, wherein the polypeptide comprises an amylase activity. In oneaspect, the amylase can be a nonsurface-active amylase. In anotheraspect, the amylase can be a surface-active amylase.

The invention provides methods for washing an object comprising thefollowing steps: (a) providing a composition comprising a polypeptidehaving an amylase activity, wherein the polypeptide comprises: apolypeptide of the invention or a polypeptide encoded by a nucleic acidof the invention; (b) providing an object; and (c) contacting thepolypeptide of step (a) and the object of step (b) under conditionswherein the composition can wash the object.

The invention provides methods for hydrolyzing starch, e.g., in a feedor a food prior to consumption by an animal, comprising the followingsteps: (a) obtaining a composition, e.g., a feed material, comprising astarch, wherein the polypeptide comprises: a polypeptide of theinvention or a polypeptide encoded by a nucleic acid of the invention;and (b) adding the polypeptide of step (a) to the composition, e.g., thefeed or food material, in an amount sufficient for a sufficient timeperiod to cause hydrolysis of the starch, thereby hydrolyzing thestarch. In one aspect, the food or feed comprises rice, corn, barley,wheat, legumes, or potato.

The invention provides methods for textile desizing comprising thefollowing steps: (a) providing a polypeptide having an amylase activity,wherein the polypeptide comprises a polypeptide of the invention or apolypeptide encoded by a nucleic acid of the invention; (b) providing afabric; and (c) contacting the polypeptide of step (a) and the fabric ofstep (b) under conditions wherein the amylase can desize the fabric.

The invention provides methods for deinking of paper or fiberscomprising the following steps: (a) providing a polypeptide having anamylase activity, wherein the polypeptide comprises a polypeptide of theinvention; (b) providing a composition comprising paper or fiber; and(c) contacting the polypeptide of step (a) and the composition of step(b) under conditions wherein the polypeptide can deink the paper orfiber.

The invention provides methods for treatment of lignocellulosic fiberscomprising the following steps: (a) providing a polypeptide having anamylase activity, wherein the polypeptide comprises a polypeptide of theinvention; (b) providing a lignocellulosic fiber; and (c) contacting thepolypeptide of step (a) and the fiber of step (b) under conditionswherein the polypeptide can treat the fiber thereby improving the fiberproperties.

The invention provides methods for producing a high-maltose or ahigh-glucose syrup comprising the following steps: (a) providing apolypeptide having an amylase activity, wherein the polypeptidecomprises a polypeptide of the invention; (b) providing a compositioncomprising a starch; and (c) contacting the polypeptide of step (a) andthe fabric of step (b) under conditions wherein the polypeptide of step(a) can liquefy the composition of step (b) thereby producing a solublestarch hydrolysate and saccharify the soluble starch hydrolysate therebyproducing the syrup. In one aspect, the starch can be from rice, corn,barley, wheat, legumes, potato, or sweet potato.

The invention provides methods for improving the flow of thestarch-containing production fluids comprising the following steps: (a)providing a polypeptide having an amylase activity, wherein thepolypeptide comprises a polypeptide of the invention; (b) providingproduction fluid; and (c) contacting the polypeptide of step (a) and theproduction fluid of step (b) under conditions wherein the amylase canhydrolyze the starch in the production fluid thereby improving its flowby decreasing its density. In one aspect, the production fluid can befrom a subterranean formation.

The invention provides anti-staling compositions comprising apolypeptide of the invention or a polypeptide encoded by a nucleic acidof the invention. The invention provides methods for preventing stalingof the baked products comprising the following steps: (a) providing apolypeptide having an amylase activity, wherein the polypeptidecomprises a polypeptide of the invention; (b) providing a compositioncontaining starch used for baking; (c) combining the polypeptide of step(a) with the composition of the step (b) under conditions wherein thepolypeptide can hydrolyze the starch in the composition used for bakingthereby preventing staling of the baked product. In one aspect, thebaked product can be bread.

The invention provides methods for using amylase in brewing or alcoholproduction comprising the following steps: (a) providing a polypeptidehaving an amylase activity, wherein the polypeptide comprises apolypeptide of the invention; (b) providing a composition containingstarch and used for brewing or in alcohol production; (c) combining thepolypeptide of step (a) with the composition of the step (b) underconditions wherein the polypeptide can hydrolyze the starch in thecomposition used for brewing or in alcohol production. In one aspect,the composition containing starch can be beer.

The invention provides methods of making a transgenic plant comprisingthe following steps: (a) introducing a heterologous nucleic acidsequence into the cell, wherein the heterologous nucleic sequencecomprises a nucleic acid sequence of the invention, thereby producing atransformed plant cell; and (b) producing a transgenic plant from thetransformed cell. In one aspect, the step (a) can further compriseintroducing the heterologous nucleic acid sequence by electroporation ormicroinjection of plant cell protoplasts. In another aspect, the step(a) can further comprise introducing the heterologous nucleic acidsequence directly to plant tissue by DNA particle bombardment.Alternatively, the step (a) can further comprise introducing theheterologous nucleic acid sequence into the plant cell DNA using anAgrobacterium tumefaciens host. In one aspect, the plant cell can be apotato, corn, rice, wheat, tobacco, or barley cell.

The invention provides methods of expressing a heterologous nucleic acidsequence in a plant cell comprising the following steps: (a)transforming the plant cell with a heterologous nucleic acid sequenceoperably linked to a promoter, wherein the heterologous nucleic sequencecomprises a nucleic acid of the invention; (b) growing the plant underconditions wherein the heterologous nucleic acids sequence is expressedin the plant cell.

The invention also provides a process for preparing a dough or a bakedproduct prepared from the dough which comprises adding an amylase of theinvention to the dough in an amount which is effective to retard thestaling of the bread. The invention also provides a dough comprisingsaid amylase and a premix comprising flour together with said amylase.Finally, the invention provides an enzymatic baking additive, whichcontains said amylase. The use of the amylase in accordance with thepresent invention provides an improved anti-staling effect as measuredby, e.g. less crumb firming, retained crumb elasticity, improvedslice-ability (e.g. fewer crumbs, non-gummy crumb), improvedpalatability or flavor.

The invention provides an isolated nucleic acid having a sequence as setforth in SEQ ID NO: 1 and variants thereof having at least 50% sequenceidentity to SEQ ID NO: 1 and encoding polypeptides having alpha amylaseactivity. One aspect of the invention is an isolated nucleic acid havinga sequence as set forth in SEQ ID NO: 1, sequences substantiallyidentical thereto, and sequences complementary thereto. Another aspectof the invention is an isolated nucleic acid including at least 10consecutive bases of a sequence as set forth in SEQ ID NO:1 nucleic acidsequences, sequences substantially identical thereto, and the sequencescomplementary thereto. In yet another aspect, the invention provides anisolated nucleic acid encoding a polypeptide having a sequence as setforth in SEQ ID NO.: 2 and variants thereof encoding a polypeptidehaving alpha amylase activity and having at least 50% sequence identityto such sequences. Another aspect of the invention is an isolatednucleic acid encoding a polypeptide or a functional fragment thereofhaving a sequence as set forth in SEQ ID NO: 2 and sequencessubstantially identical thereto. Another aspect of the invention is anisolated nucleic acid encoding a polypeptide having at least 10consecutive amino acids of a sequence as set forth in SEQ ID NO:2, andsequences substantially identical thereto. In yet another aspect, theinvention provides a purified polypeptide having a sequence as set forthin SEQ ID NO:2 and sequences substantially identical thereto. Anotheraspect of the invention is an isolated or purified antibody thatspecifically binds to a polypeptide having a sequence as set forth inSEQ ID NO:2, and sequences substantially identical thereto. Anotheraspect of the invention is an isolated or purified antibody or bindingfragment thereof, which specifically binds to a polypeptide having atleast 10 consecutive amino acids of one of the polypeptides of SEQ IDNO:2, and sequences substantially identical thereto. Another aspect ofthe invention is a method of making a polypeptide having a sequence asset forth in SEQ ID NO:2, and sequences substantially identical thereto.

The method includes introducing a nucleic acid encoding the polypeptideinto a host cell, wherein the nucleic acid is operably linked to apromoter, and culturing the host cell under conditions that allowexpression of the nucleic acid. Another aspect of the invention is amethod of making a polypeptide having at least 10 amino acids of asequence as set forth in SEQ ID NO:2, and sequences substantiallyidentical thereto. The method includes introducing a nucleic acidencoding the polypeptide into a host cell, wherein the nucleic acid isoperably linked to a promoter, and culturing the host cell underconditions that allow expression of the nucleic acid, thereby producingthe polypeptide. Another aspect of the invention is a method ofgenerating a variant including obtaining a nucleic acid having asequence as set forth in SEQ ID NO:1 nucleic acid sequences, sequencessubstantially identical thereto, sequences complementary to thesequences of SEQ ID NO:1 nucleic acid sequences, fragments comprising atleast 30 consecutive nucleotides of the foregoing sequences, andchanging one or more nucleotides in the sequence to another nucleotide,deleting one or more nucleotides in the sequence, or adding one or morenucleotides to the sequence.

Another aspect of the invention is a computer readable medium havingstored thereon a sequence as set forth in SEQ ID NO:1 nucleic acidsequences, and sequences substantially identical thereto, or apolypeptide sequence as set forth in SEQ ID NO:2, and sequencessubstantially identical thereto. Another aspect of the invention is acomputer system including a processor and a data storage device whereinthe data storage device has stored thereon a sequence as set forth inSEQ ID NO:1 nucleic acid sequences, and sequences substantiallyidentical thereto, or a polypeptide having a sequence as set forth inSEQ ID NO:2, and sequences substantially identical thereto. Anotheraspect of the invention is a method for comparing a first sequence to areference sequence wherein the first sequence is a nucleic acid having asequence as set forth in SEQ ID NO:1 nucleic acid sequences, andsequences substantially identical thereto, or a polypeptide code of SEQID NO:2, and sequences substantially identical thereto. The methodincludes reading the first sequence and the reference sequence throughuse of a computer program which compares sequences; and determiningdifferences between the first sequence and the reference sequence withthe computer program. Another aspect of the invention is a method foridentifying a feature in a sequence as set forth in SEQ ID NO:1 nucleicacid sequences, and sequences substantially identical thereto, or apolypeptide having a sequence as set forth in SEQ ID NO:2, and sequencessubstantially identical thereto, including reading the sequence throughthe use of a computer program which identifies features in sequences;and identifying features in the sequence with the computer program.Another aspect of the invention is an assay for identifying fragments orvariants of SEQ ID NO; and sequences substantially identical thereto,which retain the enzymatic function of the polypeptides of SEQ ID NO:2,and sequences substantially identical thereto. The assay includescontacting the polypeptide of SEQ ID NO:2, sequences substantiallyidentical thereto, or polypeptide fragment or variant with a substratemolecule under conditions which allow the polypeptide fragment orvariant to function, and detecting either a decrease in the level ofsubstrate or an increase in the level of the specific reaction productof the reaction between the polypeptide and substrate therebyidentifying a fragment or variant of such sequences.

The invention also provides a process for preparing a dough or a bakedproduct prepared from the dough which comprises adding an amylase of theinvention to the dough in an amount which is effective to retard thestaling of the bread. The invention also provides a dough comprisingsaid amylase and a premix comprising flour together with said amylase.Finally, the invention provides an enzymatic baking additive, whichcontains said amylase. The use of the amylase in accordance with thepresent invention provides an improved anti-staling effect as measuredby, e.g. less crumb firming, retained crumb elasticity, improvedslice-ability (e.g. fewer crumbs, non-gummy crumb), improvedpalatability or flavor.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

All publications, patents, patent applications, GenBank sequences andATCC deposits, cited herein are hereby expressly incorporated byreference for all purposes.

DESCRIPTION OF DRAWINGS

FIG. 1 is a block diagram of a computer system.

FIG. 2 is a flow diagram illustrating one aspect of a process forcomparing a new nucleotide or protein sequence with a database ofsequences in order to determine the homology levels between the newsequence and the sequences in the database.

FIG. 3 is a flow diagram illustrating one aspect of a process in acomputer for determining whether two sequences are homologous.

FIG. 4 is a flow diagram illustrating one aspect of an identifierprocess 300 for detecting the presence of a feature in a sequence.

FIG. 5 illustrates a sample Standard Curve of the assay of Example 3.

FIGS. 6A and 6B show the molecular weight fragments in syrups usingenzymes of the invention and commercial enzymes, as discussed below.

FIG. 6A shows the molecular weight fragments in syrups using enzymes ofthe invention.

FIG. 6B shows the molecular weight fragments in syrups using commercialenzymes.

FIGS. 7A-7N show sequences of the invention.

FIG. 7A shows SEQ ID NO:1 of the invention.

FIG. 7B shows SEQ ID NO:2 of the invention.

FIG. 7C shows SEQ ID NO:3 of the invention.

FIG. 7D shows SEQ ID NO:4 of the invention.

FIG. 7E shows SEQ ID NO:5 of the invention.

FIG. 7F shows SEQ ID NO:6 of the invention.

FIG. 7G shows SEQ ID NO:7 of the invention.

FIG. 7H shows SEQ ID NO:8 of the invention.

FIG. 7I shows SEQ ID NO:9 of the invention.

FIG. 7J shows SEQ ID NO:10 of the invention.

FIG. 7K shows SEQ ID NO:66 of the invention.

FIG. 7L shows SEQ ID NO:67 of the invention.

FIG. 7M shows SEQ ID NO:68 of the invention.

FIG. 7N shows SEQ ID NO:69 of the invention.

FIG. 8 shows a comparison of oligosaccharide profiles for SEQ ID NO:2and commercial amylases.

Like reference symbols in the various drawings indicate like elements.

Sequences of the invention are additionally provided in a sequencelisting.

DETAILED DESCRIPTION

The invention provides amylase enzymes, e.g., an alpha amylase,polynucleotides encoding the enzymes, methods of making and using thesepolynucleotides and polypeptides. The invention is directed to novelpolypeptides having an amylase activity, e.g., an alpha amylaseactivity, nucleic acids encoding them and antibodies that bind to them.The polypeptides of the invention can be used in a variety ofdiagnostic, therapeutic, and industrial contexts. The polypeptides ofthe invention can be used as, e.g., an additive for a detergent, forprocessing foods and for chemical synthesis utilizing a reversereaction. Additionally, the polypeptides of the invention can be used infabric treatment, alcohol production, and as additives to food or animalfeed.

In one aspect, the amylases of the invention are active at a high and/orat a low temperature, or, over a wide range of temperature. For example,they can be active in the temperatures ranging between 20° C. to 90° C.,between 30° C. to 80° C., or between 40° C. to 70° C. The invention alsoprovides amylases that have activity at alkaline pHs or at acidic pHs,e.g., low water acidity. In alternative aspects, the amylases of theinvention can have activity in acidic pHs as low as pH 5.0, pH 4.5, pH4.0, and pH 3.5. In alternative aspects, the amylases of the inventioncan have activity in alkaline pHs as high as pH 9.5, pH 10, pH 10.5, andpH 11. In one aspect, the amylases of the invention are active in thetemperature range of between about 40° C. to about 70° C. underconditions of low water activity (low water content).

The invention also provides methods for further modifying the exemplaryamylases of the invention to generate proteins with desirableproperties. For example, amylases generated by the methods of theinvention can have altered enzymatic activity, thermal stability,pH/activity profile, pH/stability profile (such as increased stabilityat low, e.g. pH<6 or pH<5, or high, e.g. pH>9, pH values), stabilitytowards oxidation, Ca²⁺ dependency, specific activity and the like. Theinvention provides for altering any property of interest. For instance,the alteration may result in a variant which, as compared to a parentenzyme, has altered enzymatic activity, or, pH or temperature activityprofiles.

In one aspect, the present invention relates to alpha amylases andpolynucleotides encoding them. As used herein, the term “alpha amylase”encompasses enzymes having alpha amylase activity, for example, enzymescapable of hydrolyzing starch to sugars. Unlike many known amylases, theexemplary amylase of the invention, set forth in SEQ ID NO:2, is not acalcium-dependent enzyme.

It is highly desirable to be able to decrease the Ca2⁺ dependency of analpha amylase. Accordingly, one aspect of the invention provides anamylase enzyme that has a decreased Ca2⁺ dependency as compared tocommercial or parent amylases. Decreased Ca2⁺ dependency will in generalhave the functional consequence that the variant exhibits a satisfactoryamylolytic activity in the presence of a lower concentration of calciumion in the extraneous medium than is necessary for a commercial orparent enzyme. It will further often have the consequence that thevariant is less sensitive to calcium ion-depleting conditions such asthose obtained in media containing calcium-complexing agents (such ascertain detergent builders).

The polynucleotides of the invention have been identified as encodingpolypeptides having alpha amylase activity. An exemplary alpha amylaseenzyme of the invention is shown in SEQ ID NO:2, also referred to hereinas SEQ ID NO:2. Such amylases of the invention are particularly usefulin corn-wet milling processes, detergents, baking processes, beveragesand in oilfields (fuel ethanol).

Alterations in properties which may be achieved in variants of theinvention are alterations in, e.g., substrate specificity, substratebinding, substrate cleavage pattern, thermal stability, pH/activityprofile, pH/stability profile, such as increased stability at low (e.g.pH<6, in particular pH<5) or high (e.g. pH>9) pH values], stabilitytowards oxidation, Ca2⁺ dependency, specific activity, and otherproperties of interest. For instance, the alteration may result in avariant which, as compared to the parent amylase, has a reduced Ca2⁺dependency and/or an altered pH/activity profile.

Corn wet milling is a process which produces corn oil, gluten meal,gluten feed and starch. Amylases of the invention, including SEQ IDNO:2, are used in the liquefaction of starch and glucoamylase is used insaccharification, producing glucose. The properties of the amylases ofthe present invention are unique in that they allow production ofliquefied syrups which can be converted to higher dextrose levels than aconventional Bacillus amylase liquefied syrup. As can be seen in FIG. 6aand FIG. 6b and in the Examples, the molecular weight profile ofliquefied starch produced by commercial amylases derived from Bacilluslicheniformis and Bacillus stearothennophilus exhibit a bimodaldistribution with a primary peak at 1000-2000 MW representingapproximately 60% of the mass with a secondary peak at 30,000-40,000 MW.In addition, there is a substantial fraction at greater than 100,000 MW.The amylases of the invention exhibit a homogeneous MW distributioncentered at 1000-2000 MW with less than 10% of the mass greater than25,000 MW. The higher MW oligosaccharides are not fully converted toglucose during saccharification. Consequently, the commercial amylaseswill contain less dextrose than saccharified syrups from theliquefaction process carried out by amylase enzymes of the presentinvention, such as that represented in SEQ ID NO:2 and functionalequivalents thereof.

Maltodextrins are utilized in a wide variety of food and coatingapplications. Amylases from Archeal sources generate an extremelyuniform maltodextrin composition (see also Leveque et al., Enzyme andMicrobial Technology 26:3-14, 2000, herein incorporated by reference).The use of the amylases of the invention to liquefy corn starch resultsin a uniform maltodextrin composition. The liquefaction can be performedat a pH of about 4.5-6.5, and preferably around pH 5.0 and attemperatures of up to 105 degrees C. or higher. In one aspect, theliquefaction is performed at a pH of 4.5.

In addition to the benefits demonstrated in saccharification, theliquefied syrups can be carbon treated, spray dried and utilized as foodadditives, thickeners, low caloric bulking agents, film forming agents,etc. It is anticipated (but not yet proven) that the homogenousmolecular weight profile maltodextrins will have performance advantagesvs. the bimodal distribution maltodextrins produced by the conventionalBacillus enzymes.

Definitions

The term “amylase” includes all polypeptides, e.g., enzymes, whichcatalyze the hydrolysis of starches. For example, an amylase activity ofthe invention includes α-amylase activity, including the ability tohydrolyze internal alpha-1,4-glucosidic linkages in starch to producesmaller molecular weight malto-dextrins. In one aspect, the α-amylaseactivity includes hydrolyzing internal alpha-1,4-glucosidic linkages instarch at random. An amylase activity of the invention includespolypeptides having glucoamylase activity, such as the ability tohydrolase glucose polymers linked by α-1,4- and α-1,6-glucosidic bonds.In one aspect, the polypeptides of the invention have glucoamylaseactivity, hydrolyzing internal α-1,4-glucosidic linkages to yieldsmaller molecular weight malto-dextrins. An amylase activity of theinvention also includes glucan 1,4-α-glucosidase activity, or,1,4-α-D-glucan glucohydrolase, commonly called glucoamylase but alsocalled amyloglucosidase and γ-amylase that, in one aspect, releasesβ-D-glucose from 1,4-α-, 1,6-α- and 1,3-α-linked glucans. An amylaseactivity of the invention also includes exo-amylase activity. An amylaseactivity of the invention also includes hydrolyzing starch at hightemperatures, low temperatures, alkaline pHs and at acidic pHs. An“amylase variant” comprises an amino acid sequence which is derived fromthe amino acid sequence of a “precursor amylase”. The precursor amylasecan include naturally-occurring amylases and recombinant amylases. Theamino acid sequence of the amylase variant can be “derived” from theprecursor amylase amino acid sequence by the substitution, deletion orinsertion of one or more amino acids of the precursor amino acidsequence. Such modification can be of the “precursor DNA sequence” whichencodes the amino acid sequence of the precursor amylase rather thanmanipulation of the precursor amylase enzyme per se. Suitable methodsfor such manipulation of the precursor DNA sequence include methodsdisclosed herein, as well as methods known to those skilled in the art.

Liquefaction” or “liquefy” means a process by which starch is convertedto shorter chain and less viscous dextrins. Generally, this processinvolves gelatinization of starch simultaneously with or followed by theaddition of alpha amylase. In commercial processes, it is preferred thatthe granular starch is derived from a source comprising corn, wheat,milo, sorghum, rye or bulgher. However, the present invention applies toany grain starch source which is useful in liquefaction, e.g., any othergrain or vegetable source known to produce starch suitable forliquefaction.

“Granular starch” or “starch granules” means a water-insoluble componentof edible grains which remains after removal of the hull, fiber,protein, fat, germ, and solubles through the steeping, mechanicalcracking, separations, screening, countercurrent rinsing andcentrifugation steps typical of the grain wet-milling process. Granularstarch comprises intact starch granules containing, almost exclusively,packed starch molecules (i.e., amylopectin and amylose). In corn, thegranular starch component comprises about 99% starch; the remaining 1%being comprised of protein, fat, ash, fiber and trace components tightlyassociated with the granules. The packing structure of granular starchseverely retards the ability of .alpha.-amylase to hydrolyze starch.Gelatinization of the starch is utilized to disrupt the granules to forma soluble starch solution and facilitate enzymatic hydrolysis.

“Starch solution” means the water soluble gelatinized starch whichresults from heating granular starch. Upon heating of the granules toabove about 72 degrees C., granular starch dissociates to form anaqueous mixture of loose starch molecules. This mixture comprising, forexample, about 75% amylopectin and 25% amylose in yellow dent corn formsa viscous solution in water. In commercial processes to form glucose orfructose, it is the starch solution which is liquefied to form a solubledextrin solution. “alpha amylase” means an enzymatic activity whichcleaves or hydrolyzes the alpha (1-4) glycosidic bond, e.g., that instarch, amylopectin or amylose polymers. Suitable alpha amylases are thenaturally occurring alpha amylases as well as recombinant or mutantamylases which are useful in liquefaction of starch. Techniques forproducing variant amylases having activity at a pH or temperature, forexample, that is different from the wild-type amylase, are includedherein.

In practicing the invention, the temperature range of the liquefactioncan be any liquefaction temperature which is known to be effective inliquefying starch. The temperature of the starch can be between about80° C. to about 115° C., or from about 100° C. to about 110° C., and/orfrom about 105° C. to about 108° C.

The term “antibody” includes a peptide or polypeptide derived from,modeled after or substantially encoded by an immunoglobulin gene orimmunoglobulin genes, or fragments thereof, capable of specificallybinding an antigen or epitope, see, e.g. Fundamental Immunology, ThirdEdition, W. E. Paul, ed., Raven Press, N.Y. (1993); Wilson (1994) J.Immunol. Methods 175:267-273; Yarmush (1992) J. Biochem. Biophys.Methods 25:85-97. The term antibody includes antigen-binding portions,i.e., “antigen binding sites,” (e.g., fragments, subsequences,complementarity determining regions (CDRs)) that retain capacity to bindantigen, including (i) a Fab fragment, a monovalent fragment consistingof the VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalentfragment comprising two Fab fragments linked by a disulfide bridge atthe hinge region; (iii) a Fd fragment consisting of the VH and CH1domains; (iv) a Fv fragment consisting of the VL and VH domains of asingle arm of an antibody, (v) a dAb fragment (Ward et al., (1989)Nature 341:544-546), which consists of a VH domain; and (vi) an isolatedcomplementarity determining region (CDR). Single chain antibodies arealso included by reference in the term “antibody.”

The terms “array” or “microarray” or “biochip” or “chip” as used hereinis a plurality of target elements, each target element comprising adefined amount of one or more polypeptides (including antibodies) ornucleic acids immobilized onto a defined area of a substrate surface, asdiscussed in further detail, below.

As used herein, the terms “computer,” “computer program” and “processor”are used in their broadest general contexts and incorporate all suchdevices, as described in detail, below. A “coding sequence of” or a“sequence encodes” a particular polypeptide or protein, is a nucleicacid sequence which is transcribed and translated into a polypeptide orprotein when placed under the control of appropriate regulatorysequences.

The term “expression cassette” as used herein refers to a nucleotidesequence which is capable of affecting expression of a structural gene(i.e., a protein coding sequence, such as an amylase of the invention)in a host compatible with such sequences. Expression cassettes includeat least a promoter operably linked with the polypeptide codingsequence; and, optionally, with other sequences, e.g., transcriptiontermination signals. Additional factors necessary or helpful ineffecting expression may also be used, e.g., enhancers. Thus, expressioncassettes also include plasmids, expression vectors, recombinantviruses, any form of recombinant “naked DNA” vector, and the like.

“Operably linked” as used herein refers to a functional relationshipbetween two or more nucleic acid (e.g., DNA) segments. Typically, itrefers to the functional relationship of transcriptional regulatorysequence to a transcribed sequence. For example, a promoter is operablylinked to a coding sequence, such as a nucleic acid of the invention, ifit stimulates or modulates the transcription of the coding sequence inan appropriate host cell or other expression system. Generally, promotertranscriptional regulatory sequences that are operably linked to atranscribed sequence are physically contiguous to the transcribedsequence, i.e., they are cis-acting. However, some transcriptionalregulatory sequences, such as enhancers, need not be physicallycontiguous or located in close proximity to the coding sequences whosetranscription they enhance.

A “vector” comprises a nucleic acid which can infect, transfect,transiently or permanently transduce a cell. It will be recognized thata vector can be a naked nucleic acid, or a nucleic acid complexed withprotein or lipid. The vector optionally comprises viral or bacterialnucleic acids and/or proteins, and/or membranes (e.g., a cell membrane,a viral lipid envelope, etc.). Vectors include, but are not limited toreplicons (e.g., RNA replicons, bacteriophages) to which fragments ofDNA may be attached and become replicated. Vectors thus include, but arenot limited to RNA, autonomous self-replicating circular or linear DNAor RNA (e.g., plasmids, viruses, and the like, see, e.g., U.S. Pat. No.5,217,879), and include both the expression and non-expression plasmids.Where a recombinant microorganism or cell culture is described ashosting an “expression vector” this includes both extra-chromosomalcircular and linear DNA and DNA that has been incorporated into the hostchromosome(s). Where a vector is being maintained by a host cell, thevector may either be stably replicated by the cells during mitosis as anautonomous structure, or is incorporated within the host's genome.

As used herein, the term “promoter” includes all sequences capable ofdriving transcription of a coding sequence in a cell, e.g., a plantcell. Thus, promoters used in the constructs of the invention includecis-acting transcriptional control elements and regulatory sequencesthat are involved in regulating or modulating the timing and/or rate oftranscription of a gene. For example, a promoter can be a cis-actingtranscriptional control element, including an enhancer, a promoter, atranscription terminator, an origin of replication, a chromosomalintegration sequence, 5′ and 3′ untranslated regions, or an intronicsequence, which are involved in transcriptional regulation. Thesecis-acting sequences typically interact with proteins or otherbiomolecules to carry out (turn on/off, regulate, modulate, etc.)transcription. “Constitutive” promoters are those that drive expressioncontinuously under most environmental conditions and states ofdevelopment or cell differentiation. “Inducible” or “regulatable”promoters direct expression of the nucleic acid of the invention underthe influence of environmental conditions or developmental conditions.Examples of environmental conditions that may affect transcription byinducible promoters include anaerobic conditions, elevated temperature,drought, or the presence of light.

“Tissue-specific” promoters are transcriptional control elements thatare only active in particular cells or tissues or organs, e.g., inplants or animals. Tissue-specific regulation may be achieved by certainintrinsic factors which ensure that genes encoding proteins specific toa given tissue are expressed. Such factors are known to exist in mammalsand plants so as to allow for specific tissues to develop.

The term “plant” includes whole plants, plant parts (e.g., leaves,stems, flowers, roots, etc.), plant protoplasts, seeds and plant cellsand progeny of same. The class of plants which can be used in the methodof the invention is generally as broad as the class of higher plantsamenable to transformation techniques, including angiosperms(monocotyledonous and dicotyledonous plants), as well as gymnosperms. Itincludes plants of a variety of ploidy levels, including polyploid,diploid, haploid and hemizygous states. As used herein, the term“transgenic plant” includes plants or plant cells into which aheterologous nucleic acid sequence has been inserted, e.g., the nucleicacids and various recombinant constructs (e.g., expression cassettes) ofthe invention.

“Plasmids” can be commercially available, publicly available on anunrestricted basis, or can be constructed from available plasmids inaccord with published procedures. Equivalent plasmids to those describedherein are known in the art and will be apparent to the ordinarilyskilled artisan.

The term “gene” includes a nucleic acid sequence comprising a segment ofDNA involved in producing a transcription product (e.g., a message),which in turn is translated to produce a polypeptide chain, or regulatesgene transcription, reproduction or stability. Genes can include regionspreceding and following the coding region, such as leader and trailer,promoters and enhancers, as well as, where applicable, interveningsequences (introns) between individual coding segments (exons).

The phrases “nucleic acid” or “nucleic acid sequence” includesoligonucleotide, nucleotide, polynucleotide, or to a fragment of any ofthese, to DNA or RNA (e.g., mRNA, rRNA, tRNA) of genomic or syntheticorigin which may be single-stranded or double-stranded and may representa sense or antisense strand, to peptide nucleic acid (PNA), or to anyDNA-like or RNA-like material, natural or synthetic in origin,including, e.g., iRNA, ribonucleoproteins (e.g., iRNPs). The termencompasses nucleic acids, i.e., oligonucleotides, containing knownanalogues of natural nucleotides. The term also encompassesnucleic-acid-like structures with synthetic backbones, see e.g., Mata(1997) Toxicol. Appl. Pharmacol. 144:189-197; Strauss-Soukup (1997)Biochemistry 36:8692-8698; Samstag (1996) Antisense Nucleic Acid DrugDev 6:153-156.

“Amino acid” or “amino acid sequence” include an oligopeptide, peptide,polypeptide, or protein sequence, or to a fragment, portion, or subunitof any of these, and to naturally occurring or synthetic molecules. Theterms “polypeptide” and “protein” include amino acids joined to eachother by peptide bonds or modified peptide bonds, i.e., peptideisosteres, and may contain modified amino acids other than the 20gene-encoded amino acids. The term “polypeptide” also includes peptidesand polypeptide fragments, motifs and the like. The term also includesglycosylated polypeptides. The peptides and polypeptides of theinvention also include all “mimetic” and “peptidomimetic” forms, asdescribed in further detail, below.

The term “isolated” includes a material removed from its originalenvironment, e.g., the natural environment if it is naturally occurring.For example, a naturally occurring polynucleotide or polypeptide presentin a living animal is not isolated, but the same polynucleotide orpolypeptide, separated from some or all of the coexisting materials inthe natural system, is isolated. Such polynucleotides could be part of avector and/or such polynucleotides or polypeptides could be part of acomposition, and still be isolated in that such vector or composition isnot part of its natural environment. As used herein, an isolatedmaterial or composition can also be a “purified” composition, i.e., itdoes not require absolute purity; rather, it is intended as a relativedefinition. Individual nucleic acids obtained from a library can beconventionally purified to electrophoretic homogeneity. In alternativeaspects, the invention provides nucleic acids which have been purifiedfrom genomic DNA or from other sequences in a library or otherenvironment by at least one, two, three, four, five or more orders ofmagnitude.

As used herein, the term “recombinant” can include nucleic acidsadjacent to a “backbone” nucleic acid to which it is not adjacent in itsnatural environment. In one aspect, nucleic acids represent 5% or moreof the number of nucleic acid inserts in a population of nucleic acid“backbone molecules.” “Backbone molecules” according to the inventioninclude nucleic acids such as expression vectors, self-replicatingnucleic acids, viruses, integrating nucleic acids, and other vectors ornucleic acids used to maintain or manipulate a nucleic acid insert ofinterest. In one aspect, the enriched nucleic acids represent 10%, 15%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or more of the numberof nucleic acid inserts in the population of recombinant backbonemolecules. “Recombinant” polypeptides or proteins refer to polypeptidesor proteins produced by recombinant DNA techniques; e.g., produced fromcells transformed by an exogenous DNA construct encoding the desiredpolypeptide or protein. “Synthetic” polypeptides or protein are thoseprepared by chemical synthesis, as described in further detail, below.

A promoter sequence can be “operably linked to” a coding sequence whenRNA polymerase which initiates transcription at the promoter willtranscribe the coding sequence into mRNA, as discussed further, below.

“Oligonucleotide” includes either a single stranded polydeoxynucleotideor two complementary polydeoxynucleotide strands which may be chemicallysynthesized. Such synthetic oligonucleotides have no 5′ phosphate andthus will not ligate to another oligonucleotide without adding aphosphate with an ATP in the presence of a kinase. A syntheticoligonucleotide can ligate to a fragment that has not beendephosphorylated.

The phrase “substantially identical” in the context of two nucleic acidsor polypeptides, can refer to two or more sequences that have, e.g., atleast about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%,62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more nucleotide oramino acid residue (sequence) identity, when compared and aligned formaximum correspondence, as measured using one any known sequencecomparison algorithm, as discussed in detail below, or by visualinspection. In alternative aspects, the invention provides nucleic acidand polypeptide sequences having substantial identity to an exemplarysequence of the invention, e.g., SEQ ID NO:1, SEQ ID NO:2, over a regionof at least about 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400,450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 or moreresidues, or a region ranging from between about 50 residues to the fulllength of the nucleic acid or polypeptide. Nucleic acid sequences of theinvention can be substantially identical over the entire length of apolypeptide coding region.

A “substantially identical” amino acid sequence also can include asequence that differs from a reference sequence by one or moreconservative or non-conservative amino acid substitutions, deletions, orinsertions, particularly when such a substitution occurs at a site thatis not the active site of the molecule, and provided that thepolypeptide essentially retains its functional properties. Aconservative amino acid substitution, for example, substitutes one aminoacid for another of the same class (e.g., substitution of onehydrophobic amino acid, such as isoleucine, valine, leucine, ormethionine, for another, or substitution of one polar amino acid foranother, such as substitution of arginine for lysine, glutamic acid foraspartic acid or glutamine for asparagine). One or more amino acids canbe deleted, for example, from an amylase, resulting in modification ofthe structure of the polypeptide, without significantly altering itsbiological activity. For example, amino- or carboxyl-terminal aminoacids that are not required for amylase activity can be removed.

“Hybridization” includes the process by which a nucleic acid strandjoins with a complementary strand through base pairing. Hybridizationreactions can be sensitive and selective so that a particular sequenceof interest can be identified even in samples in which it is present atlow concentrations. Stringent conditions can be defined by, for example,the concentrations of salt or formamide in the prehybridization andhybridization solutions, or by the hybridization temperature, and arewell known in the art. For example, stringency can be increased byreducing the concentration of salt, increasing the concentration offormamide, or raising the hybridization temperature, altering the timeof hybridization, as described in detail, below. In alternative aspects,nucleic acids of the invention are defined by their ability to hybridizeunder various stringency conditions (e.g., high, medium, and low), asset forth herein.

“Variant” includes polynucleotides or polypeptides of the inventionmodified at one or more base pairs, codons, introns, exons, or aminoacid residues (respectively) yet still retain the biological activity ofan amylase of the invention. Variants can be produced by any number ofmeans included methods such as, for example, error-prone PCR, shuffling,oligonucleotide-directed mutagenesis, assembly PCR, sexual PCRmutagenesis, in vivo mutagenesis, cassette mutagenesis, recursiveensemble mutagenesis, exponential ensemble mutagenesis, site-specificmutagenesis, gene reassembly, GSSM and any combination thereof.Techniques for producing variant amylase having activity at a pH ortemperature, for example, that is different from a wild-type amylase,are included herein.

The term “saturation mutagenesis” or “GSSM” includes a method that usesdegenerate oligonucleotide primers to introduce point mutations into apolynucleotide, as described in detail, below.

The term “optimized directed evolution system” or “optimized directedevolution” includes a method for reassembling fragments of relatednucleic acid sequences, e.g., related genes, and explained in detail,below.

The term “synthetic ligation reassembly” or “SLR” includes a method ofligating oligonucleotide fragments in a non-stochastic fashion, andexplained in detail, below.

Generating and Manipulating Nucleic Acids

The invention provides nucleic acids, including expression cassettessuch as expression vectors, encoding the polypeptides of the invention.The invention also includes methods for discovering new amylasesequences using the nucleic acids of the invention. The invention alsoincludes methods for inhibiting the expression of amylase genes,transcripts and polypeptides using the nucleic acids of the invention.Also provided are methods for modifying the nucleic acids of theinvention by, e.g., synthetic ligation reassembly, optimized directedevolution system and/or saturation mutagenesis.

The nucleic acids of the invention can be made, isolated and/ormanipulated by, e.g., cloning and expression of cDNA libraries,amplification of message or genomic DNA by PCR, and the like. Inpracticing the methods of the invention, homologous genes can bemodified by manipulating a template nucleic acid, as described herein.The invention can be practiced in conjunction with any method orprotocol or device known in the art, which are well described in thescientific and patent literature.

General Techniques

The nucleic acids used to practice this invention, whether RNA, iRNA,antisense nucleic acid, cDNA, genomic DNA, vectors, viruses or hybridsthereof, may be isolated from a variety of sources, geneticallyengineered, amplified, and/or expressed/generated recombinantly.Recombinant polypeptides generated from these nucleic acids can beindividually isolated or cloned and tested for a desired activity. Anyrecombinant expression system can be used, including bacterial,mammalian, yeast, insect or plant cell expression systems.

Alternatively, these nucleic acids can be synthesized in vitro bywell-known chemical synthesis techniques, as described in, e.g., Adams(1983) J. Am. Chem. Soc. 105:661; Belousov (1997) Nucleic Acids Res.25:3440-3444; Frenkel (1995) Free Radic. Biol. Med. 19:373-380; Blommers(1994) Biochemistry 33:7886-7896; Narang (1979) Meth. Enzymol. 68:90;Brown (1979) Meth. Enzymol. 68:109; Beaucage (1981) Tetra. Lett.22:1859; U.S. Pat. No. 4,458,066.

Techniques for the manipulation of nucleic acids, such as, e.g.,subcloning, labeling probes (e.g., random-primer labeling using Klenowpolymerase, nick translation, amplification), sequencing, hybridizationand the like are well described in the scientific and patent literature,see, e.g., Sambrook, ed., MOLECULAR CLONING: A LABORATORY MANUAL (2NDED.), Vols. 1-3, Cold Spring Harbor Laboratory, (1989); CURRENTPROTOCOLS IN MOLECULAR BIOLOGY, Ausubel, ed. John Wiley & Sons, Inc.,New York (1997); LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULARBIOLOGY: HYBRIDIZATION WITH NUCLEIC ACID PROBES, Part I. Theory andNucleic Acid Preparation, Tijssen, ed. Elsevier, N.Y. (1993).

Another useful means of obtaining and manipulating nucleic acids used topractice the methods of the invention is to clone from genomic samples,and, if desired, screen and re-clone inserts isolated or amplified from,e.g., genomic clones or cDNA clones. Sources of nucleic acid used in themethods of the invention include genomic or cDNA libraries contained in,e.g., mammalian artificial chromosomes (MACs), see, e.g., U.S. Pat. Nos.5,721,118; 6,025,155; human artificial chromosomes, see, e.g., Rosenfeld(1997) Nat. Genet. 15:333-335; yeast artificial chromosomes (YAC);bacterial artificial chromosomes (BAC); P1 artificial chromosomes, see,e.g., Woon (1998) Genomics 50:306-316; P1-derived vectors (PACs), see,e.g., Kern (1997) Biotechniques 23:120-124; cosmids, recombinantviruses, phages or plasmids.

In one aspect, a nucleic acid encoding a polypeptide of the invention isassembled in appropriate phase with a leader sequence capable ofdirecting secretion of the translated polypeptide or fragment thereof.

The invention provides fusion proteins and nucleic acids encoding them.A polypeptide of the invention can be fused to a heterologous peptide orpolypeptide, such as N-terminal identification peptides which impartdesired characteristics, such as increased stability or simplifiedpurification. Peptides and polypeptides of the invention can also besynthesized and expressed as fusion proteins with one or more additionaldomains linked thereto for, e.g., producing a more immunogenic peptide,to more readily isolate a recombinantly synthesized peptide, to identifyand isolate antibodies and antibody-expressing B cells, and the like.Detection and purification facilitating domains include, e.g., metalchelating peptides such as polyhistidine tracts and histidine-tryptophanmodules that allow purification on immobilized metals, protein A domainsthat allow purification on immobilized immunoglobulin, and the domainutilized in the FLAGS extension/affinity purification system (ImmunexCorp, Seattle Wash.). The inclusion of a cleavable linker sequences suchas Factor Xa or enterokinase (Invitrogen, San Diego Calif.) between apurification domain and the motif-comprising peptide or polypeptide tofacilitate purification. For example, an expression vector can includean epitope-encoding nucleic acid sequence linked to six histidineresidues followed by a thioredoxin and an enterokinase cleavage site(see e.g., Williams (1995) Biochemistry 34:1787-1797; Dobeli (1998)Protein Expr. Purif. 12:404-414). The histidine residues facilitatedetection and purification while the enterokinase cleavage site providesa means for purifying the epitope from the remainder of the fusionprotein. Technology pertaining to vectors encoding fusion proteins andapplication of fusion proteins are well described in the scientific andpatent literature, see e.g., Kroll (1993) DNA Cell. Biol., 12:441-53.

Transcriptional and Translational Control Sequences

The invention provides nucleic acid (e.g., DNA) sequences of theinvention operatively linked to expression (e.g., transcriptional ortranslational) control sequence(s), e.g., promoters or enhancers, todirect or modulate RNA synthesis/expression. The expression controlsequence can be in an expression vector. Exemplary bacterial promotersinclude lad, lacZ, T3, T7, gpt, lambda PR, PL and trp. Exemplaryeukaryotic promoters include CMV immediate early, HSV thymidine kinase,early and late SV40, LTRs from retrovirus, and mouse metallothionein I.

Promoters suitable for expressing a polypeptide in bacteria include theE. coli lac or trp promoters, the lad promoter, the lacZ promoter, theT3 promoter, the T7 promoter, the gpt promoter, the lambda PR promoter,the lambda PL promoter, promoters from operons encoding glycolyticenzymes such as 3-phosphoglycerate kinase (PGK), and the acidphosphatase promoter. Eukaryotic promoters include the CMV immediateearly promoter, the HSV thymidine kinase promoter, heat shock promoters,the early and late SV40 promoter, LTRs from retroviruses, and the mousemetallothionein-I promoter. Other promoters known to control expressionof genes in prokaryotic or eukaryotic cells or their viruses may also beused.

Tissue-Specific Plant Promoters

The invention provides expression cassettes that can be expressed in atissue-specific manner, e.g., that can express an amylase of theinvention in a tissue-specific manner. The invention also providesplants or seeds that express an amylase of the invention in atissue-specific manner. The tissue-specificity can be seed specific,stem specific, leaf specific, root specific, fruit specific and thelike.

In one aspect, a constitutive promoter such as the CaMV 35S promoter canbe used for expression in specific parts of the plant or seed orthroughout the plant. For example, for overexpression, a plant promoterfragment can be employed which will direct expression of a nucleic acidin some or all tissues of a plant, e.g., a regenerated plant. Suchpromoters are referred to herein as “constitutive” promoters and areactive under most environmental conditions and states of development orcell differentiation. Examples of constitutive promoters include thecauliflower mosaic virus (CaMV) 35S transcription initiation region, the1′- or 2′-promoter derived from T-DNA of Agrobacterium tumefaciens, andother transcription initiation regions from various plant genes known tothose of skill. Such genes include, e.g., ACT11 from Arabidopsis (Huang(1996) Plant Mol. Biol. 33:125-139); Cat3 from Arabidopsis (GenBank No.U43147, Zhong (1996) Mol. Gen. Genet. 251:196-203); the gene encodingstearoyl-acyl carrier protein desaturase from Brassica napus (GenbankNo. X74782, Solocombe (1994) Plant Physiol. 104:1167-1176); GPc1 frommaize (GenBank No. X15596; Martinez (1989) J. Mol. Biol 208:551-565);the Gpc2 from maize (GenBank No. U45855, Manjunath (1997) Plant Mol.Biol. 33:97-112); plant promoters described in U.S. Pat. Nos. 4,962,028;5,633,440.

The invention uses tissue-specific or constitutive promoters derivedfrom viruses which can include, e.g., the tobamovirus subgenomicpromoter (Kumagai (1995) Proc. Natl. Acad. Sci. USA 92:1679-1683; therice tungro bacilliform virus (RTBV), which replicates only in phloemcells in infected rice plants, with its promoter which drives strongphloem-specific reporter gene expression; the cassava vein mosaic virus(CVMV) promoter, with highest activity in vascular elements, in leafmesophyll cells, and in root tips (Verdaguer (1996) Plant Mol. Biol.31:1129-1139).

Alternatively, the plant promoter may direct expression ofamylase-expressing nucleic acid in a specific tissue, organ or cell type(i.e. tissue-specific promoters) or may be otherwise under more preciseenvironmental or developmental control or under the control of aninducible promoter. Examples of environmental conditions that may affecttranscription include anaerobic conditions, elevated temperature, thepresence of light, or sprayed with chemicals/hormones. For example, theinvention incorporates the drought-inducible promoter of maize (Busk(1997) supra); the cold, drought, and high salt inducible promoter frompotato (Kirch (1997) Plant Mol. Biol. 33:897 909).

Tissue-specific promoters can promote transcription only within acertain time frame of developmental stage within that tissue. See, e.g.,Blazquez (1998) Plant Cell 10:791-800, characterizing the ArabidopsisLEAFY gene promoter. See also Cardon (1997) Plant J 12:367-77,describing the transcription factor SPL3, which recognizes a conservedsequence motif in the promoter region of the A. thaliana floral meristemidentity gene AP1; and Mandel (1995) Plant Molecular Biology, Vol. 29,pp 995-1004, describing the meristem promoter eIF4. Tissue specificpromoters which are active throughout the life cycle of a particulartissue can be used. In one aspect, the nucleic acids of the inventionare operably linked to a promoter active primarily only in cotton fibercells. In one aspect, the nucleic acids of the invention are operablylinked to a promoter active primarily during the stages of cotton fibercell elongation, e.g., as described by Rinehart (1996) supra. Thenucleic acids can be operably linked to the Fbl2A gene promoter to bepreferentially expressed in cotton fiber cells (Ibid). See also, John(1997) Proc. Natl. Acad. Sci. USA 89:5769-5773; John, et al., U.S. Pat.Nos. 5,608,148 and 5,602,321, describing cotton fiber-specific promotersand methods for the construction of transgenic cotton plants.Root-specific promoters may also be used to express the nucleic acids ofthe invention. Examples of root-specific promoters include the promoterfrom the alcohol dehydrogenase gene (DeLisle (1990) Int. Rev. Cytol.123:39-60). Other promoters that can be used to express the nucleicacids of the invention include, e.g., ovule-specific, embryo-specific,endosperm-specific, integument-specific, seed coat-specific promoters,or some combination thereof; a leaf-specific promoter (see, e.g., Busk(1997) Plant J. 11:1285 1295, describing a leaf-specific promoter inmaize); the ORF13 promoter from Agrobacterium rhizogenes (which exhibitshigh activity in roots, see, e.g., Hansen (1997) supra); a maize pollenspecific promoter (see, e.g., Guerrero (1990) Mol. Gen. Genet. 224:161168); a tomato promoter active during fruit ripening, senescence andabscission of leaves and, to a lesser extent, of flowers can be used(see, e.g., Blume (1997) Plant J. 12:731 746); a pistil-specificpromoter from the potato SK2 gene (see, e.g., Ficker (1997) Plant Mol.Biol. 35:425 431); the Blec4 gene from pea, which is active in epidermaltissue of vegetative and floral shoot apices of transgenic alfalfamaking it a useful tool to target the expression of foreign genes to theepidermal layer of actively growing shoots or fibers; the ovule-specificBEL1 gene (see, e.g., Reiser (1995) Cell 83:735-742, GenBank No.U39944); and/or, the promoter in Klee, U.S. Pat. No. 5,589,583,describing a plant promoter region is capable of conferring high levelsof transcription in meristematic tissue and/or rapidly dividing cells.

Alternatively, plant promoters which are inducible upon exposure toplant hormones, such as auxins, are used to express the nucleic acids ofthe invention. For example, the invention can use the auxin-responseelements E1 promoter fragment (AuxREs) in the soybean (Glycine max L.)(Liu (1997) Plant Physiol. 115:397-407); the auxin-responsiveArabidopsis GST6 promoter (also responsive to salicylic acid andhydrogen peroxide) (Chen (1996) Plant J. 10: 955-966); theauxin-inducible parC promoter from tobacco (Sakai (1996) 37:906-913); aplant biotin response element (Streit (1997) Mol. Plant MicrobeInteract. 10:933-937); and, the promoter responsive to the stresshormone abscisic acid (Sheen (1996) Science 274:1900-1902).

The nucleic acids of the invention can also be operably linked to plantpromoters which are inducible upon exposure to chemicals reagents whichcan be applied to the plant, such as herbicides or antibiotics. Forexample, the maize In2-2 promoter, activated by benzenesulfonamideherbicide safeners, can be used (De Veylder (1997) Plant Cell Physiol.38:568-577); application of different herbicide safeners inducesdistinct gene expression patterns, including expression in the root,hydathodes, and the shoot apical meristem. Coding sequence can be underthe control of, e.g., a tetracycline-inducible promoter, e.g., asdescribed with transgenic tobacco plants containing the Avena sativa L.(oat) arginine decarboxylase gene (Masgrau (1997) Plant J. 11:465-473);or, a salicylic acid-responsive element (Stange (1997) Plant J.11:1315-1324). Using chemically- (e.g., hormone- or pesticide-) inducedpromoters, i.e., promoter responsive to a chemical which can be appliedto the transgenic plant in the field, expression of a polypeptide of theinvention can be induced at a particular stage of development of theplant. Thus, the invention also provides for transgenic plantscontaining an inducible gene encoding for polypeptides of the inventionwhose host range is limited to target plant species, such as corn, rice,barley, wheat, potato or other crops, inducible at any stage ofdevelopment of the crop.

One of skill will recognize that a tissue-specific plant promoter maydrive expression of operably linked sequences in tissues other than thetarget tissue. Thus, a tissue-specific promoter is one that drivesexpression preferentially in the target tissue or cell type, but mayalso lead to some expression in other tissues as well.

The nucleic acids of the invention can also be operably linked to plantpromoters which are inducible upon exposure to chemicals reagents. Thesereagents include, e.g., herbicides, synthetic auxins, or antibioticswhich can be applied, e.g., sprayed, onto transgenic plants. Inducibleexpression of the amylase-producing nucleic acids of the invention willallow the grower to select plants with the optimal starch/sugar ratio.The development of plant parts can thus controlled. In this way theinvention provides the means to facilitate the harvesting of plants andplant parts. For example, in various embodiments, the maize In2-2promoter, activated by benzenesulfonamide herbicide safeners, is used(De Veylder (1997) Plant Cell Physiol. 38:568-577); application ofdifferent herbicide safeners induces distinct gene expression patterns,including expression in the root, hydathodes, and the shoot apicalmeristem. Coding sequences of the invention are also under the controlof a tetracycline-inducible promoter, e.g., as described with transgenictobacco plants containing the Avena sativa L. (oat) argininedecarboxylase gene (Masgrau (1997) Plant J. 11:465-473); or, a salicylicacid-responsive element (Stange (1997) Plant J. 11:1315-1324).

If proper polypeptide expression is desired, a polyadenylation region atthe 3′-end of the coding region should be included. The polyadenylationregion can be derived from the natural gene, from a variety of otherplant genes, or from genes in the Agrobacterial T-DNA.

Expression Vectors and Cloning Vehicles

The invention provides expression vectors and cloning vehiclescomprising nucleic acids of the invention, e.g., sequences encoding theamylases of the invention. Expression vectors and cloning vehicles ofthe invention can comprise viral particles, baculovirus, phage,plasmids, phagemids, cosmids, fosmids, bacterial artificial chromosomes,viral DNA (e.g., vaccinia, adenovirus, foul pox virus, pseudorabies andderivatives of SV40), P1-based artificial chromosomes, yeast plasmids,yeast artificial chromosomes, and any other vectors specific forspecific hosts of interest (such as bacillus, Aspergillus and yeast).Vectors of the invention can include chromosomal, non-chromosomal andsynthetic DNA sequences. Large numbers of suitable vectors are known tothose of skill in the art, and are commercially available. Exemplaryvectors are include: bacterial: pQE vectors (Qiagen), pBluescriptplasmids, pNH vectors, (lambda-ZAP vectors (Stratagene); ptrc99a,pKK223-3, pDR540, pRIT2T (Pharmacia); Eukaryotic: pXT1, pSG5(Stratagene), pSVK3, pBPV, pMSG, pSVLSV40 (Pharmacia). However, anyother plasmid or other vector may be used so long as they are replicableand viable in the host. Low copy number or high copy number vectors maybe employed with the present invention.

The expression vector can comprise a promoter, a ribosome binding sitefor translation initiation and a transcription terminator. The vectormay also include appropriate sequences for amplifying expression.Mammalian expression vectors can comprise an origin of replication, anynecessary ribosome binding sites, a polyadenylation site, splice donorand acceptor sites, transcriptional termination sequences, and 5′flanking non-transcribed sequences. In some aspects, DNA sequencesderived from the SV40 splice and polyadenylation sites may be used toprovide the required non-transcribed genetic elements. In one aspect,the expression vectors contain one or more selectable marker genes topermit selection of host cells containing the vector. Such selectablemarkers include genes encoding dihydrofolate reductase or genesconferring neomycin resistance for eukaryotic cell culture, genesconferring tetracycline or ampicillin resistance in E. coli, and the S.cerevisiae TRP1 gene. Promoter regions can be selected from any desiredgene using chloramphenicol transferase (CAT) vectors or other vectorswith selectable markers.

Vectors for expressing the polypeptide or fragment thereof in eukaryoticcells can also contain enhancers to increase expression levels.Enhancers are cis-acting elements of DNA, usually from about 10 to about300 bp in length that act on a promoter to increase its transcription.Examples include the SV40 enhancer on the late side of the replicationorigin by 100 to 270, the cytomegalovirus early promoter enhancer, thepolyoma enhancer on the late side of the replication origin, and theadenovirus enhancers.

A nucleic acid sequence can be inserted into a vector by a variety ofprocedures. In general, the sequence is ligated to the desired positionin the vector following digestion of the insert and the vector withappropriate restriction endonucleases. Alternatively, blunt ends in boththe insert and the vector may be ligated. A variety of cloningtechniques are known in the art, e.g., as described in Ausubel andSambrook. Such procedures and others are deemed to be within the scopeof those skilled in the art.

The vector can be in the form of a plasmid, a viral particle, or aphage. Other vectors include chromosomal, non-chromosomal and syntheticDNA sequences, derivatives of SV40; bacterial plasmids, phage DNA,baculovirus, yeast plasmids, vectors derived from combinations ofplasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl poxvirus, and pseudorabies. A variety of cloning and expression vectors foruse with prokaryotic and eukaryotic hosts are described by, e.g.,Sambrook.

Particular bacterial vectors which can be used include the commerciallyavailable plasmids comprising genetic elements of the well known cloningvector pBR322 (ATCC 37017), pKK223-3 (Pharmacia Fine Chemicals, Uppsala,Sweden), GEM1 (Promega Biotec, Madison, Wis., USA) pQE70, pQE60, pQE-9(Qiagen), pD10, psiX174 pBluescript II KS, pNH8A, pNH16a, pNH18A, pNH46A(Stratagene), ptrc99a, pKK223-3, pKK233-3, DR540, pRIT5 (Pharmacia),pKK232-8 and pCM7. Particular eukaryotic vectors include pSV2CAT, pOG44,pXT1, pSG (Stratagene) pSVK3, pBPV, pMSG, and pSVL (Pharmacia). However,any other vector may be used as long as it is replicable and viable inthe host cell.

The nucleic acids of the invention can be expressed in expressioncassettes, vectors or viruses and transiently or stably expressed inplant cells and seeds. One exemplary transient expression system usesepisomal expression systems, e.g., cauliflower mosaic virus (CaMV) viralRNA generated in the nucleus by transcription of an episomalmini-chromosome containing supercoiled DNA, see, e.g., Covey (1990)Proc. Natl. Acad. Sci. USA 87:1633-1637. Alternatively, codingsequences, i.e., all or sub-fragments of sequences of the invention canbe inserted into a plant host cell genome becoming an integral part ofthe host chromosomal DNA. Sense or antisense transcripts can beexpressed in this manner. A vector comprising the sequences (e.g.,promoters or coding regions) from nucleic acids of the invention cancomprise a marker gene that confers a selectable phenotype on a plantcell or a seed. For example, the marker may encode biocide resistance,particularly antibiotic resistance, such as resistance to kanamycin,G418, bleomycin, hygromycin, or herbicide resistance, such as resistanceto chlorosulfuron or Basta.

Expression vectors capable of expressing nucleic acids and proteins inplants are well known in the art, and can include, e.g., vectors fromAgrobacterium spp., potato virus X (see, e.g., Angell (1997) EMBO J.16:3675-3684), tobacco mosaic virus (see, e.g., Casper (1996) Gene173:69-73), tomato bushy stunt virus (see, e.g., Hillman (1989) Virology169:42-50), tobacco etch virus (see, e.g., Dolja (1997) Virology234:243-252), bean golden mosaic virus (see, e.g., Morinaga (1993)Microbiol Immunol. 37:471-476), cauliflower mosaic virus (see, e.g.,Cecchini (1997) Mol. Plant Microbe Interact. 10:1094-1101), maize Ac/Dstransposable element (see, e.g., Rubin (1997) Mol. Cell. Biol.17:6294-6302; Kunze (1996) Curr. Top. Microbiol. Immunol. 204:161-194),and the maize suppressor-mutator (Spm) transposable element (see, e.g.,Schlappi (1996) Plant Mol. Biol. 32:717-725); and derivatives thereof.

In one aspect, the expression vector can have two replication systems toallow it to be maintained in two organisms, for example in mammalian orinsect cells for expression and in a prokaryotic host for cloning andamplification. Furthermore, for integrating expression vectors, theexpression vector can contain at least one sequence homologous to thehost cell genome. It can contain two homologous sequences which flankthe expression construct. The integrating vector can be directed to aspecific locus in the host cell by selecting the appropriate homologoussequence for inclusion in the vector. Constructs for integrating vectorsare well known in the art.

Expression vectors of the invention may also include a selectable markergene to allow for the selection of bacterial strains that have beentransformed, e.g., genes which render the bacteria resistant to drugssuch as ampicillin, chloramphenicol, erythromycin, kanamycin, neomycinand tetracycline. Selectable markers can also include biosyntheticgenes, such as those in the histidine, tryptophan and leucinebiosynthetic pathways.

Host Cells and Transformed Cells

The invention also provides a transformed cell comprising a nucleic acidsequence of the invention, e.g., a sequence encoding an amylase of theinvention, or a vector of the invention. The host cell may be any of thehost cells familiar to those skilled in the art, including prokaryoticcells, eukaryotic cells, such as bacterial cells, fungal cells, yeastcells, mammalian cells, insect cells, or plant cells. Exemplarybacterial cells include E. coli, Streptomyces, Bacillus subtilis,Salmonella typhimurium and various species within the generaPseudomonas, Streptomyces, and Staphylococcus. Exemplary insect cellsinclude Drosophila S2 and Spodoptera Sf9. Exemplary animal cells includeCHO, COS or Bowes melanoma or any mouse or human cell line. Theselection of an appropriate host is within the abilities of thoseskilled in the art. Techniques for transforming a wide variety of higherplant species are well known and described in the technical andscientific literature. See, e.g., Weising (1988) Ann. Rev. Genet.22:421-477, U.S. Pat. No. 5,750,870.

The vector can be introduced into the host cells using any of a varietyof techniques, including transformation, transfection, transduction,viral infection, gene guns, or Ti-mediated gene transfer. Particularmethods include calcium phosphate transfection, DEAE-Dextran mediatedtransfection, lipofection, or electroporation (Davis, L., Dibner, M.,Battey, I., Basic Methods in Molecular Biology, (1986)).

In one aspect, the nucleic acids or vectors of the invention areintroduced into the cells for screening, thus, the nucleic acids enterthe cells in a manner suitable for subsequent expression of the nucleicacid. The method of introduction is largely dictated by the targetedcell type. Exemplary methods include CaPO₄ precipitation, liposomefusion, lipofection (e.g., LIPOFECTIN™), electroporation, viralinfection, etc. The candidate nucleic acids may stably integrate intothe genome of the host cell (for example, with retroviral introduction)or may exist either transiently or stably in the cytoplasm (i.e. throughthe use of traditional plasmids, utilizing standard regulatorysequences, selection markers, etc.). As many pharmaceutically importantscreens require human or model mammalian cell targets, retroviralvectors capable of transfecting such targets are preferred.

Where appropriate, the engineered host cells can be cultured inconventional nutrient media modified as appropriate for activatingpromoters, selecting transformants or amplifying the genes of theinvention. Following transformation of a suitable host strain and growthof the host strain to an appropriate cell density, the selected promotermay be induced by appropriate means (e.g., temperature shift or chemicalinduction) and the cells may be cultured for an additional period toallow them to produce the desired polypeptide or fragment thereof.

Cells can be harvested by centrifugation, disrupted by physical orchemical means, and the resulting crude extract is retained for furtherpurification. Microbial cells employed for expression of proteins can bedisrupted by any convenient method, including freeze-thaw cycling,sonication, mechanical disruption, or use of cell lysing agents. Suchmethods are well known to those skilled in the art. The expressedpolypeptide or fragment thereof can be recovered and purified fromrecombinant cell cultures by methods including ammonium sulfate orethanol precipitation, acid extraction, anion or cation exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography, affinity chromatography, hydroxylapatite chromatographyand lectin chromatography. Protein refolding steps can be used, asnecessary, in completing configuration of the polypeptide. If desired,high performance liquid chromatography (HPLC) can be employed for finalpurification steps.

Various mammalian cell culture systems can also be employed to expressrecombinant protein. Examples of mammalian expression systems includethe COS-7 lines of monkey kidney fibroblasts and other cell linescapable of expressing proteins from a compatible vector, such as theC127, 3T3, CHO, HeLa and BHK cell lines.

The constructs in host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence. Dependingupon the host employed in a recombinant production procedure, thepolypeptides produced by host cells containing the vector may beglycosylated or may be non-glycosylated. Polypeptides of the inventionmay or may not also include an initial methionine amino acid residue.

Cell-free translation systems can also be employed to produce apolypeptide of the invention. Cell-free translation systems can usemRNAs transcribed from a DNA construct comprising a promoter operablylinked to a nucleic acid encoding the polypeptide or fragment thereof.In some aspects, the DNA construct may be linearized prior to conductingan in vitro transcription reaction. The transcribed mRNA is thenincubated with an appropriate cell-free translation extract, such as arabbit reticulocyte extract, to produce the desired polypeptide orfragment thereof.

The expression vectors can contain one or more selectable marker genesto provide a phenotypic trait for selection of transformed host cellssuch as dihydrofolate reductase or neomycin resistance for eukaryoticcell culture, or such as tetracycline or ampicillin resistance in E.coli.

Amplification of Nucleic Acids

In practicing the invention, nucleic acids of the invention and nucleicacids encoding the polypeptides of the invention, or modified nucleicacids of the invention, can be reproduced by amplification.Amplification can also be used to clone or modify the nucleic acids ofthe invention. Thus, the invention provides amplification primersequence pairs for amplifying nucleic acids of the invention. One ofskill in the art can design amplification primer sequence pairs for anypart of or the full length of these sequences.

Amplification reactions can also be used to quantify the amount ofnucleic acid in a sample (such as the amount of message in a cellsample), label the nucleic acid (e.g., to apply it to an array or ablot), detect the nucleic acid, or quantify the amount of a specificnucleic acid in a sample. In one aspect of the invention, messageisolated from a cell or a cDNA library are amplified.

The skilled artisan can select and design suitable oligonucleotideamplification primers. Amplification methods are also well known in theart, and include, e.g., polymerase chain reaction, PCR (see, e.g., PCRPROTOCOLS, A GUIDE TO METHODS AND APPLICATIONS, ed. Innis, AcademicPress, N.Y. (1990) and PCR STRATEGIES (1995), ed. Innis, Academic Press,Inc., N.Y., ligase chain reaction (LCR) (see, e.g., Wu (1989) Genomics4:560; Landegren (1988) Science 241:1077; Barringer (1990) Gene 89:117);transcription amplification (see, e.g., Kwoh (1989) Proc. Natl. Acad.Sci. USA 86:1173); and, self-sustained sequence replication (see, e.g.,Guatelli (1990) Proc. Natl. Acad. Sci. USA 87:1874); Q Beta replicaseamplification (see, e.g., Smith (1997) J. Clin. Microbiol.35:1477-1491), automated Q-beta replicase amplification assay (see,e.g., Burg (1996) Mol. Cell. Probes 10:257-271) and other RNA polymerasemediated techniques (e.g., NASBA, Cangene, Mississauga, Ontario); seealso Berger (1987) Methods Enzymol. 152:307-316; Sambrook; Ausubel; U.S.Pat. Nos. 4,683,195 and 4,683,202; Sooknanan (1995) Biotechnology13:563-564.

Determining the Degree of Sequence Identity

The invention provides nucleic acids comprising sequences having atleast about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%,62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete(100%) sequence identity to an exemplary nucleic acid of the inventionover a region of at least about 50, 75, 100, 150, 200, 250, 300, 350,400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050,1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550 or more,residues. The invention provides polypeptides comprising sequenceshaving at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%,60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, orcomplete (100%) sequence identity to an exemplary polypeptide of theinvention. The extent of sequence identity (homology) may be determinedusing any computer program and associated parameters, including thosedescribed herein, such as BLAST 2.2.2. or FASTA version 3.0t78, with thedefault parameters.

Homologous sequences also include RNA sequences in which uridinesreplace the thymines in the nucleic acid sequences. The homologoussequences may be obtained using any of the procedures described hereinor may result from the correction of a sequencing error. It will beappreciated that the nucleic acid sequences as set forth herein can berepresented in the traditional single character format (see, e.g.,Stryer, Lubert. Biochemistry, 3rd Ed., W. H Freeman & Co., New York) orin any other format which records the identity of the nucleotides in asequence.

Various sequence comparison programs identified herein are used in thisaspect of the invention. Protein and/or nucleic acid sequence identities(homologies) may be evaluated using any of the variety of sequencecomparison algorithms and programs known in the art. Such algorithms andprograms include, but are not limited to, TBLASTN, BLASTP, FASTA,TFASTA, and CLUSTALW (Pearson and Lipman, Proc. Natl. Acad. Sci. USA85(8):2444-2448, 1988; Altschul et al., J. Mol. Biol. 215(3):403-410,1990; Thompson et al., Nucleic Acids Res. 22(2):4673-4680, 1994; Higginset al., Methods Enzymol. 266:383-402, 1996; Altschul et al., J. Mol.Biol. 215(3):403-410, 1990; Altschul et al., Nature Genetics 3:266-272,1993).

Homology or identity can be measured using sequence analysis software(e.g., Sequence Analysis Software Package of the Genetics ComputerGroup, University of Wisconsin Biotechnology Center, 1710 UniversityAvenue, Madison, Wis. 53705). Such software matches similar sequences byassigning degrees of homology to various deletions, substitutions andother modifications. The terms “homology” and “identity” in the contextof two or more nucleic acids or polypeptide sequences, refer to two ormore sequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same whencompared and aligned for maximum correspondence over a comparison windowor designated region as measured using any number of sequence comparisonalgorithms or by manual alignment and visual inspection. For sequencecomparison, one sequence can act as a reference sequence, e.g., asequence of the invention, to which test sequences are compared. Whenusing a sequence comparison algorithm, test and reference sequences areentered into a computer, subsequence coordinates are designated, ifnecessary, and sequence algorithm program parameters are designated.Default program parameters can be used, or alternative parameters can bedesignated. The sequence comparison algorithm then calculates thepercent sequence identities for the test sequences relative to thereference sequence, based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the numbers of contiguous residues. For example, inalternative aspects of the invention, contiguous residues ranginganywhere from 20 to the full length of an exemplary polypeptide ornucleic acid sequence of the invention are compared to a referencesequence of the same number of contiguous positions after the twosequences are optimally aligned. If the reference sequence has therequisite sequence identity to an exemplary polypeptide or nucleic acidsequence of the invention, e.g., 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ormore sequence identity to a sequence of the invention, that sequence iswithin the scope of the invention. In alternative embodiments,subsequences ranging from about 20 to 600, about 50 to 200, and about100 to 150 are compared to a reference sequence of the same number ofcontiguous positions after the two sequences are optimally aligned.Methods of alignment of sequence for comparison are well known in theart. Optimal alignment of sequences for comparison can be conducted,e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl.Math. 2:482, 1981, by the homology alignment algorithm of Needleman &Wunsch, J. Mol. Biol. 48:443, 1970, by the search for similarity methodof person & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444, 1988, bycomputerized implementations of these algorithms (GAP, BESTFIT, FASTA,and TFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup, 575 Science Dr., Madison, Wis.), or by manual alignment andvisual inspection. Other algorithms for determining homology or identityinclude, for example, in addition to a BLAST program (Basic LocalAlignment Search Tool at the National Center for BiologicalInformation), ALIGN, AMAS (Analysis of Multiply Aligned Sequences), AMPS(Protein Multiple Sequence Alignment), ASSET (Aligned SegmentStatistical Evaluation Tool), BANDS, BESTSCOR, BIOSCAN (BiologicalSequence Comparative Analysis Node), BLIMPS (BLocks IMProved Searcher),FASTA, Intervals & Points, BMB, CLUSTAL V, CLUSTAL W, CONSENSUS,LCONSENSUS, WCONSENSUS, Smith-Waterman algorithm, DARWIN, Las Vegasalgorithm, FNAT (Forced Nucleotide Alignment Tool), Framealign,Framesearch, DYNAMIC, FILTER, FSAP (Fristensky Sequence AnalysisPackage), GAP (Global Alignment Program), GENAL, GIBBS, GenQuest, ISSC(Sensitive Sequence Comparison), LALIGN (Local Sequence Alignment), LCP(Local Content Program), MACAW (Multiple Alignment Construction &Analysis Workbench), MAP (Multiple Alignment Program), MBLKP, MBLKN,PIMA (Pattern-Induced Multi-sequence Alignment), SAGA (SequenceAlignment by Genetic Algorithm) and WHAT-IF. Such alignment programs canalso be used to screen genome databases to identify polynucleotidesequences having substantially identical sequences. A number of genomedatabases are available, for example, a substantial portion of the humangenome is available as part of the Human Genome Sequencing Project(Gibbs, 1995). Several genomes have been sequenced, e.g., M. genitalium(Fraser et al., 1995), M. jannaschii (Bult et al., 1996), H. influenzae(Fleischmann et al., 1995), E. coli (Blattner et al., 1997), and yeast(S. cerevisiae) (Mewes et al., 1997), and D. melanogaster (Adams et al.,2000). Significant progress has also been made in sequencing the genomesof model organism, such as mouse, C. elegans, and Arabadopsis sp.Databases containing genomic information annotated with some functionalinformation are maintained by different organization, and are accessiblevia the internet.

BLAST, BLAST 2.0 and BLAST 2.2.2 algorithms are also used to practicethe invention. They are described, e.g., in Altschul (1977) Nuc. AcidsRes. 25:3389-3402; Altschul (1990) J. Mol. Biol. 215:403-410. Softwarefor performing BLAST analyses is publicly available through the NationalCenter for Biotechnology Information. This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold (Altschul (1990) supra). These initial neighborhoodword hits act as seeds for initiating searches to find longer HSPscontaining them. The word hits are extended in both directions alongeach sequence for as far as the cumulative alignment score can beincreased. Cumulative scores are calculated using, for nucleotidesequences, the parameters M (reward score for a pair of matchingresidues; always >0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectations (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915)alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands. The BLAST algorithm also performs a statisticalanalysis of the similarity between two sequences (see, e.g., Karlin &Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873). One measure ofsimilarity provided by BLAST algorithm is the smallest sum probability(P(N)), which provides an indication of the probability by which a matchbetween two nucleotide or amino acid sequences would occur by chance.For example, a nucleic acid is considered similar to a referencessequence if the smallest sum probability in a comparison of the testnucleic acid to the reference nucleic acid is less than about 0.2, morepreferably less than about 0.01, and most preferably less than about0.001. In one aspect, protein and nucleic acid sequence homologies areevaluated using the Basic Local Alignment Search Tool (“BLAST”). Forexample, five specific BLAST programs can be used to perform thefollowing task: (1) BLASTP and BLAST3 compare an amino acid querysequence against a protein sequence database; (2) BLASTN compares anucleotide query sequence against a nucleotide sequence database; (3)BLASTX compares the six-frame conceptual translation products of a querynucleotide sequence (both strands) against a protein sequence database;(4) TBLASTN compares a query protein sequence against a nucleotidesequence database translated in all six reading frames (both strands);and, (5) TBLASTX compares the six-frame translations of a nucleotidequery sequence against the six-frame translations of a nucleotidesequence database. The BLAST programs identify homologous sequences byidentifying similar segments, which are referred to herein as“high-scoring segment pairs,” between a query amino or nucleic acidsequence and a test sequence which is preferably obtained from a proteinor nucleic acid sequence database. High-scoring segment pairs arepreferably identified (i.e., aligned) by means of a scoring matrix, manyof which are known in the art. Preferably, the scoring matrix used isthe BLOSUM62 matrix (Gonnet et al., Science 256:1443-1445, 1992;Henikoff and Henikoff, Proteins 17:49-61, 1993). Less preferably, thePAM or PAM250 matrices may also be used (see, e.g., Schwartz andDayhoff, eds., 1978, Matrices for Detecting Distance Relationships:Atlas of Protein Sequence and Structure, Washington: National BiomedicalResearch Foundation).

In one aspect of the invention, to determine if a nucleic acid has therequisite sequence identity to be within the scope of the invention, theNCBI BLAST 2.2.2 programs is used, default options to blastp. There areabout 38 setting options in the BLAST 2.2.2 program. In this exemplaryaspect of the invention, all default values are used except for thedefault filtering setting (i.e., all parameters set to default exceptfiltering which is set to OFF); in its place a “-F F” setting is used,which disables filtering. Use of default filtering often results inKarlin-Altschul violations due to short length of sequence.

The default values used in this exemplary aspect of the inventioninclude:

-   -   “Filter for low complexity: ON    -   Word Size: 3    -   Matrix: Blosum62    -   Gap Costs: Existence: 11    -   Extension: 1”

Other default settings can be: filter for low complexity OFF, word sizeof 3 for protein, BLOSUM62 matrix, gap existence penalty of −11 and agap extension penalty of −1. An exemplary NCBI BLAST 2.2.2 programsetting has the “−W” option default to 0. This means that, if not set,the word size defaults to 3 for proteins and 11 for nucleotides.

Computer Systems and Computer Program Products

To determine and identify sequence identities, structural homologies,motifs and the like in silico, the sequence of the invention can bestored, recorded, and manipulated on any medium which can be read andaccessed by a computer. Accordingly, the invention provides computers,computer systems, computer readable mediums, computer programs productsand the like recorded or stored thereon the nucleic acid and polypeptidesequences of the invention. As used herein, the words “recorded” and“stored” refer to a process for storing information on a computermedium. A skilled artisan can readily adopt any known methods forrecording information on a computer readable medium to generatemanufactures comprising one or more of the nucleic acid and/orpolypeptide sequences of the invention.

Another aspect of the invention is a computer readable medium havingrecorded thereon at least one nucleic acid and/or polypeptide sequenceof the invention. Computer readable media include magnetically readablemedia, optically readable media, electronically readable media andmagnetic/optical media. For example, the computer readable media may bea hard disk, a floppy disk, a magnetic tape, CD-ROM, Digital VersatileDisk (DVD), Random Access Memory (RAM), or Read Only Memory (ROM) aswell as other types of other media known to those skilled in the art.

Aspects of the invention include systems (e.g., internet based systems),particularly computer systems, which store and manipulate the sequencesand sequence information described herein. One example of a computersystem 100 is illustrated in block diagram form in FIG. 1. As usedherein, “a computer system” refers to the hardware components, softwarecomponents, and data storage components used to analyze a nucleotide orpolypeptide sequence of the invention. The computer system 100 caninclude a processor for processing, accessing and manipulating thesequence data. The processor 105 can be any well-known type of centralprocessing unit, such as, for example, the Pentium III from IntelCorporation, or similar processor from Sun, Motorola, Compaq, AMD orInternational Business Machines. The computer system 100 is a generalpurpose system that comprises the processor 105 and one or more internaldata storage components 110 for storing data, and one or more dataretrieving devices for retrieving the data stored on the data storagecomponents. A skilled artisan can readily appreciate that any one of thecurrently available computer systems are suitable.

In one aspect, the computer system 100 includes a processor 105connected to a bus which is connected to a main memory 115 (preferablyimplemented as RAM) and one or more internal data storage devices 110,such as a hard drive and/or other computer readable media having datarecorded thereon. The computer system 100 can further include one ormore data retrieving device 118 for reading the data stored on theinternal data storage devices 110. The data retrieving device 118 mayrepresent, for example, a floppy disk drive, a compact disk drive, amagnetic tape drive, or a modem capable of connection to a remote datastorage system (e.g., via the internet) etc. In some embodiments, theinternal data storage device 110 is a removable computer readable mediumsuch as a floppy disk, a compact disk, a magnetic tape, etc. containingcontrol logic and/or data recorded thereon. The computer system 100 mayadvantageously include or be programmed by appropriate software forreading the control logic and/or the data from the data storagecomponent once inserted in the data retrieving device. The computersystem 100 includes a display 120 which is used to display output to acomputer user. It should also be noted that the computer system 100 canbe linked to other computer systems 125 a-c in a network or wide areanetwork to provide centralized access to the computer system 100.Software for accessing and processing the nucleotide or amino acidsequences of the invention can reside in main memory 115 duringexecution. In some aspects, the computer system 100 may further comprisea sequence comparison algorithm for comparing a nucleic acid sequence ofthe invention. The algorithm and sequence(s) can be stored on a computerreadable medium. A “sequence comparison algorithm” refers to one or moreprograms which are implemented (locally or remotely) on the computersystem 100 to compare a nucleotide sequence with other nucleotidesequences and/or compounds stored within a data storage means. Forexample, the sequence comparison algorithm may compare the nucleotidesequences of the invention stored on a computer readable medium toreference sequences stored on a computer readable medium to identifyhomologies or structural motifs.

The parameters used with the above algorithms may be adapted dependingon the sequence length and degree of homology studied. In some aspects,the parameters may be the default parameters used by the algorithms inthe absence of instructions from the user. FIG. 2 is a flow diagramillustrating one aspect of a process 200 for comparing a new nucleotideor protein sequence with a database of sequences in order to determinethe homology levels between the new sequence and the sequences in thedatabase. The database of sequences can be a private database storedwithin the computer system 100, or a public database such as GENBANKthat is available through the Internet. The process 200 begins at astart state 201 and then moves to a state 202 wherein the new sequenceto be compared is stored to a memory in a computer system 100. Asdiscussed above, the memory could be any type of memory, including RAMor an internal storage device. The process 200 then moves to a state 204wherein a database of sequences is opened for analysis and comparison.The process 200 then moves to a state 206 wherein the first sequencestored in the database is read into a memory on the computer. Acomparison is then performed at a state 210 to determine if the firstsequence is the same as the second sequence. It is important to notethat this step is not limited to performing an exact comparison betweenthe new sequence and the first sequence in the database. Well-knownmethods are known to those of skill in the art for comparing twonucleotide or protein sequences, even if they are not identical. Forexample, gaps can be introduced into one sequence in order to raise thehomology level between the two tested sequences. The parameters thatcontrol whether gaps or other features are introduced into a sequenceduring comparison are normally entered by the user of the computersystem. Once a comparison of the two sequences has been performed at thestate 210, a determination is made at a decision state 210 whether thetwo sequences are the same. Of course, the term “same” is not limited tosequences that are absolutely identical. Sequences that are within thehomology parameters entered by the user will be marked as “same” in theprocess 200. If a determination is made that the two sequences are thesame, the process 200 moves to a state 214 wherein the name of thesequence from the database is displayed to the user. This state notifiesthe user that the sequence with the displayed name fulfills the homologyconstraints that were entered. Once the name of the stored sequence isdisplayed to the user, the process 200 moves to a decision state 218wherein a determination is made whether more sequences exist in thedatabase. If no more sequences exist in the database, then the process200 terminates at an end state 220. However, if more sequences do existin the database, then the process 200 moves to a state 224 wherein apointer is moved to the next sequence in the database so that it can becompared to the new sequence. In this manner, the new sequence isaligned and compared with every sequence in the database. It should benoted that if a determination had been made at the decision state 212that the sequences were not homologous, then the process 200 would moveimmediately to the decision state 218 in order to determine if any othersequences were available in the database for comparison. Accordingly,one aspect of the invention is a computer system comprising a processor,a data storage device having stored thereon a nucleic acid sequence ofthe invention and a sequence comparer for conducting the comparison. Thesequence comparer may indicate a homology level between the sequencescompared or identify structural motifs, or it may identify structuralmotifs in sequences which are compared to these nucleic acid codes andpolypeptide codes. FIG. 3 is a flow diagram illustrating one embodimentof a process 250 in a computer for determining whether two sequences arehomologous. The process 250 begins at a start state 252 and then movesto a state 254 wherein a first sequence to be compared is stored to amemory. The second sequence to be compared is then stored to a memory ata state 256. The process 250 then moves to a state 260 wherein the firstcharacter in the first sequence is read and then to a state 262 whereinthe first character of the second sequence is read. It should beunderstood that if the sequence is a nucleotide sequence, then thecharacter would normally be either A, T, C, G or U. If the sequence is aprotein sequence, then it can be a single letter amino acid code so thatthe first and sequence sequences can be easily compared. A determinationis then made at a decision state 264 whether the two characters are thesame. If they are the same, then the process 250 moves to a state 268wherein the next characters in the first and second sequences are read.A determination is then made whether the next characters are the same.If they are, then the process 250 continues this loop until twocharacters are not the same. If a determination is made that the nexttwo characters are not the same, the process 250 moves to a decisionstate 274 to determine whether there are any more characters eithersequence to read. If there are not any more characters to read, then theprocess 250 moves to a state 276 wherein the level of homology betweenthe first and second sequences is displayed to the user. The level ofhomology is determined by calculating the proportion of charactersbetween the sequences that were the same out of the total number ofsequences in the first sequence. Thus, if every character in a first 100nucleotide sequence aligned with an every character in a secondsequence, the homology level would be 100%.

Alternatively, the computer program can compare a reference sequence toa sequence of the invention to determine whether the sequences differ atone or more positions. The program can record the length and identity ofinserted, deleted or substituted nucleotides or amino acid residues withrespect to the sequence of either the reference or the invention. Thecomputer program may be a program which determines whether a referencesequence contains a single nucleotide polymorphism (SNP) with respect toa sequence of the invention, or, whether a sequence of the inventioncomprises a SNP of a known sequence. Thus, in some aspects, the computerprogram is a program which identifies SNPs. The method may beimplemented by the computer systems described above and the methodillustrated in FIG. 3. The method can be performed by reading a sequenceof the invention and the reference sequences through the use of thecomputer program and identifying differences with the computer program.

In other aspects the computer based system comprises an identifier foridentifying features within a nucleic acid or polypeptide of theinvention. An “identifier” refers to one or more programs whichidentifies certain features within a nucleic acid sequence. For example,an identifier may comprise a program which identifies an open readingframe (ORF) in a nucleic acid sequence. FIG. 4 is a flow diagramillustrating one aspect of an identifier process 300 for detecting thepresence of a feature in a sequence. The process 300 begins at a startstate 302 and then moves to a state 304 wherein a first sequence that isto be checked for features is stored to a memory 115 in the computersystem 100. The process 300 then moves to a state 306 wherein a databaseof sequence features is opened. Such a database would include a list ofeach feature's attributes along with the name of the feature. Forexample, a feature name could be “Initiation Codon” and the attributewould be “ATG”. Another example would be the feature name “TAATAA Box”and the feature attribute would be “TAATAA”. An example of such adatabase is produced by the University of Wisconsin Genetics ComputerGroup. Alternatively, the features may be structural polypeptide motifssuch as alpha helices, beta sheets, or functional polypeptide motifssuch as enzymatic active sites, helix-turn-helix motifs or other motifsknown to those skilled in the art. Once the database of features isopened at the state 306, the process 300 moves to a state 308 whereinthe first feature is read from the database. A comparison of theattribute of the first feature with the first sequence is then made at astate 310. A determination is then made at a decision state 316 whetherthe attribute of the feature was found in the first sequence. If theattribute was found, then the process 300 moves to a state 318 whereinthe name of the found feature is displayed to the user. The process 300then moves to a decision state 320 wherein a determination is madewhether move features exist in the database. If no more features doexist, then the process 300 terminates at an end state 324. However, ifmore features do exist in the database, then the process 300 reads thenext sequence feature at a state 326 and loops back to the state 310wherein the attribute of the next feature is compared against the firstsequence. If the feature attribute is not found in the first sequence atthe decision state 316, the process 300 moves directly to the decisionstate 320 in order to determine if any more features exist in thedatabase. Thus, in one aspect, the invention provides a computer programthat identifies open reading frames (ORFs).

A polypeptide or nucleic acid sequence of the invention can be storedand manipulated in a variety of data processor programs in a variety offormats. For example, a sequence can be stored as text in a wordprocessing file, such as MicrosoftWORD or WORDPERFECT or as an ASCIIfile in a variety of database programs familiar to those of skill in theart, such as DB2, SYBASE, or ORACLE. In addition, many computer programsand databases may be used as sequence comparison algorithms,identifiers, or sources of reference nucleotide sequences or polypeptidesequences to be compared to a nucleic acid sequence of the invention.The programs and databases used to practice the invention include, butare not limited to: MacPattern (EMBL), DiscoveryBase (MolecularApplications Group), GeneMine (Molecular Applications Group), Look(Molecular Applications Group), MacLook (Molecular Applications Group),BLAST and BLAST2 (NCBI), BLASTN and BLASTX (Altschul et al, J. Mol.Biol. 215: 403, 1990), FASTA (Pearson and Lipman, Proc. Natl. Acad. Sci.USA, 85: 2444, 1988), FASTDB (Brutlag et al. Comp. App. Biosci.6:237-245, 1990), Catalyst (Molecular Simulations Inc.), Catalyst/SHAPE(Molecular Simulations Inc.), Cerius2.DBAccess (Molecular SimulationsInc.), HypoGen (Molecular Simulations Inc.), Insight II, (MolecularSimulations Inc.), Discover (Molecular Simulations Inc.), CHARMm(Molecular Simulations Inc.), Felix (Molecular Simulations Inc.),DelPhi, (Molecular Simulations Inc.), QuanteMM, (Molecular SimulationsInc.), Homology (Molecular Simulations Inc.), Modeler (MolecularSimulations Inc.), ISIS (Molecular Simulations Inc.), Quanta/ProteinDesign (Molecular Simulations Inc.), WebLab (Molecular SimulationsInc.), WebLab Diversity Explorer (Molecular Simulations Inc.), GeneExplorer (Molecular Simulations Inc.), SeqFold (Molecular SimulationsInc.), the MDL Available Chemicals Directory database, the MDL Drug DataReport data base, the Comprehensive Medicinal Chemistry database,Derwent's World Drug Index database, the BioByteMasterFile database, theGenbank database, and the Genseqn database. Many other programs and databases would be apparent to one of skill in the art given the presentdisclosure.

Motifs which may be detected using the above programs include sequencesencoding leucine zippers, helix-turn-helix motifs, glycosylation sites,ubiquitination sites, alpha helices, and beta sheets, signal sequencesencoding signal peptides which direct the secretion of the encodedproteins, sequences implicated in transcription regulation such ashomeoboxes, acidic stretches, enzymatic active sites, substrate bindingsites, and enzymatic cleavage sites.

Hybridization of Nucleic Acids

The invention provides isolated or recombinant nucleic acids thathybridize under stringent conditions to an exemplary sequence of theinvention, or a nucleic acid that encodes a polypeptide of theinvention. The stringent conditions can be highly stringent conditions,medium stringent conditions, low stringent conditions, including thehigh and reduced stringency conditions described herein. In one aspect,it is the stringency of the wash conditions that set forth theconditions which determine whether a nucleic acid is within the scope ofthe invention, as discussed below.

In alternative embodiments, nucleic acids of the invention as defined bytheir ability to hybridize under stringent conditions can be betweenabout five residues and the full length of nucleic acid of theinvention; e.g., they can be at least 5, 10, 15, 20, 25, 30, 35, 40, 50,55, 60, 65, 70, 75, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500,550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, or more, residues inlength. Nucleic acids shorter than full length are also included. Thesenucleic acids can be useful as, e.g., hybridization probes, labelingprobes, PCR oligonucleotide probes, iRNA, antisense or sequencesencoding antibody binding peptides (epitopes), motifs, active sites andthe like.

In one aspect, nucleic acids of the invention are defined by theirability to hybridize under high stringency comprises conditions of about50% formamide at about 37° C. to 42° C. In one aspect, nucleic acids ofthe invention are defined by their ability to hybridize under reducedstringency comprising conditions in about 35% to 25% formamide at about30° C. to 35° C.

Alternatively, nucleic acids of the invention are defined by theirability to hybridize under high stringency comprising conditions at 42°C. in 50% formamide, 5×SSPE, 0.3% SDS, and a repetitive sequenceblocking nucleic acid, such as cot-1 or salmon sperm DNA (e.g., 200 n/mlsheared and denatured salmon sperm DNA). In one aspect, nucleic acids ofthe invention are defined by their ability to hybridize under reducedstringency conditions comprising 35% formamide at a reduced temperatureof 35° C.

Following hybridization, the filter may be washed with 6×SSC, 0.5% SDSat 50° C. These conditions are considered to be “moderate” conditionsabove 25% formamide and “low” conditions below 25% formamide. A specificexample of “moderate” hybridization conditions is when the abovehybridization is conducted at 30% formamide. A specific example of “lowstringency” hybridization conditions is when the above hybridization isconducted at 10% formamide.

The temperature range corresponding to a particular level of stringencycan be further narrowed by calculating the purine to pyrimidine ratio ofthe nucleic acid of interest and adjusting the temperature accordingly.Nucleic acids of the invention are also defined by their ability tohybridize under high, medium, and low stringency conditions as set forthin Ausubel and Sambrook. Variations on the above ranges and conditionsare well known in the art. Hybridization conditions are discussedfurther, below.

The above procedure may be modified to identify nucleic acids havingdecreasing levels of homology to the probe sequence. For example, toobtain nucleic acids of decreasing homology to the detectable probe,less stringent conditions may be used. For example, the hybridizationtemperature may be decreased in increments of 5° C. from 68° C. to 42°C. in a hybridization buffer having a Na⁺ concentration of approximately1M. Following hybridization, the filter may be washed with 2×SSC, 0.5%SDS at the temperature of hybridization. These conditions are consideredto be “moderate” conditions above 50° C. and “low” conditions below 50°C. A specific example of “moderate” hybridization conditions is when theabove hybridization is conducted at 55° C. A specific example of “lowstringency” hybridization conditions is when the above hybridization isconducted at 45° C.

Alternatively, the hybridization may be carried out in buffers, such as6×SSC, containing formamide at a temperature of 42° C. In this case, theconcentration of formamide in the hybridization buffer may be reduced in5% increments from 50% to 0% to identify clones having decreasing levelsof homology to the probe. Following hybridization, the filter may bewashed with 6×SSC, 0.5% SDS at 50° C. These conditions are considered tobe “moderate” conditions above 25% formamide and “low” conditions below25% formamide. A specific example of “moderate” hybridization conditionsis when the above hybridization is conducted at 30% formamide. Aspecific example of “low stringency” hybridization conditions is whenthe above hybridization is conducted at 10% formamide.

However, the selection of a hybridization format is not critical—it isthe stringency of the wash conditions that set forth the conditionswhich determine whether a nucleic acid is within the scope of theinvention. Wash conditions used to identify nucleic acids within thescope of the invention include, e.g.: a salt concentration of about 0.02molar at pH 7 and a temperature of at least about 50° C. or about 55° C.to about 60° C.; or, a salt concentration of about 0.15 M NaCl at 72° C.for about 15 minutes; or, a salt concentration of about 0.2×SSC at atemperature of at least about 50° C. or about 55° C. to about 60° C. forabout 15 to about 20 minutes; or, the hybridization complex is washedtwice with a solution with a salt concentration of about 2×SSCcontaining 0.1% SDS at room temperature for 15 minutes and then washedtwice by 0.1×SSC containing 0.1% SDS at 68° C. for 15 minutes; or,equivalent conditions. See Sambrook, Tijssen and Ausubel for adescription of SSC buffer and equivalent conditions.

These methods may be used to isolate nucleic acids of the invention.

Oligonucleotides Probes and Methods for Using them

The invention also provides nucleic acid probes that can be used, e.g.,for identifying nucleic acids encoding a polypeptide with an amylaseactivity or fragments thereof or for identifying amylase genes. In oneaspect, the probe comprises at least 10 consecutive bases of a nucleicacid of the invention. Alternatively, a probe of the invention can be atleast about 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70,80, 90, 100, 110, 120, 130, 150 or about 10 to 50, about 20 to 60 about30 to 70, consecutive bases of a sequence as set forth in a nucleic acidof the invention. The probes identify a nucleic acid by binding and/orhybridization. The probes can be used in arrays of the invention, seediscussion below, including, e.g., capillary arrays. The probes of theinvention can also be used to isolate other nucleic acids orpolypeptides.

The probes of the invention can be used to determine whether abiological sample, such as a soil sample, contains an organism having anucleic acid sequence of the invention or an organism from which thenucleic acid was obtained. In such procedures, a biological samplepotentially harboring the organism from which the nucleic acid wasisolated is obtained and nucleic acids are obtained from the sample. Thenucleic acids are contacted with the probe under conditions which permitthe probe to specifically hybridize to any complementary sequencespresent in the sample. Where necessary, conditions which permit theprobe to specifically hybridize to complementary sequences may bedetermined by placing the probe in contact with complementary sequencesfrom samples known to contain the complementary sequence, as well ascontrol sequences which do not contain the complementary sequence.Hybridization conditions, such as the salt concentration of thehybridization buffer, the formamide concentration of the hybridizationbuffer, or the hybridization temperature, may be varied to identifyconditions which allow the probe to hybridize specifically tocomplementary nucleic acids (see discussion on specific hybridizationconditions).

If the sample contains the organism from which the nucleic acid wasisolated, specific hybridization of the probe is then detected.Hybridization may be detected by labeling the probe with a detectableagent such as a radioactive isotope, a fluorescent dye or an enzymecapable of catalyzing the formation of a detectable product. Manymethods for using the labeled probes to detect the presence ofcomplementary nucleic acids in a sample are familiar to those skilled inthe art. These include Southern Blots, Northern Blots, colonyhybridization procedures, and dot blots. Protocols for each of theseprocedures are provided in Ausubel and Sambrook.

Alternatively, more than one probe (at least one of which is capable ofspecifically hybridizing to any complementary sequences which arepresent in the nucleic acid sample), may be used in an amplificationreaction to determine whether the sample contains an organism containinga nucleic acid sequence of the invention (e.g., an organism from whichthe nucleic acid was isolated). In one aspect, the probes compriseoligonucleotides. In one aspect, the amplification reaction may comprisea PCR reaction. PCR protocols are described in Ausubel and Sambrook (seediscussion on amplification reactions). In such procedures, the nucleicacids in the sample are contacted with the probes, the amplificationreaction is performed, and any resulting amplification product isdetected. The amplification product may be detected by performing gelelectrophoresis on the reaction products and staining the gel with anintercalator such as ethidium bromide. Alternatively, one or more of theprobes may be labeled with a radioactive isotope and the presence of aradioactive amplification product may be detected by autoradiographyafter gel electrophoresis.

Probes derived from sequences near the 3′ or 5′ ends of a nucleic acidsequence of the invention can also be used in chromosome walkingprocedures to identify clones containing additional, e.g., genomicsequences. Such methods allow the isolation of genes which encodeadditional proteins of interest from the host organism.

In one aspect, nucleic acid sequences of the invention are used asprobes to identify and isolate related nucleic acids. In some aspects,the so-identified related nucleic acids may be cDNAs or genomic DNAsfrom organisms other than the one from which the nucleic acid of theinvention was first isolated. In such procedures, a nucleic acid sampleis contacted with the probe under conditions which permit the probe tospecifically hybridize to related sequences. Hybridization of the probeto nucleic acids from the related organism is then detected using any ofthe methods described above.

In nucleic acid hybridization reactions, the conditions used to achievea particular level of stringency can vary, depending on the nature ofthe nucleic acids being hybridized. For example, the length, degree ofcomplementarity, nucleotide sequence composition (e.g., GC v. ATcontent), and nucleic acid type (e.g., RNA v. DNA) of the hybridizingregions of the nucleic acids can be considered in selectinghybridization conditions. An additional consideration is whether one ofthe nucleic acids is immobilized, for example, on a filter.Hybridization can be carried out under conditions of low stringency,moderate stringency or high stringency. As an example of nucleic acidhybridization, a polymer membrane containing immobilized denaturednucleic acids is first prehybridized for 30 minutes at 45° C. in asolution consisting of 0.9 M NaCl, 50 mM NaH₂PO4, pH 7.0, 5.0 mMNa₂EDTA, 0.5% SDS, 10×Denhardt's, and 0.5 mg/ml polyriboadenylic acid.Approximately 2×10⁷ cpm (specific activity 4-9×10⁸ cpm/ug) of ³²Pend-labeled oligonucleotide probe can then added to the solution. After12-16 hours of incubation, the membrane is washed for 30 minutes at roomtemperature (RT) in 1×SET (150 mM NaCl, 20 mM Tris hydrochloride, pH7.8, 1 mM Na₂EDTA) containing 0.5% SDS, followed by a 30 minute wash infresh 1×SET at Tm-10° C. for the oligonucleotide probe. The membrane isthen exposed to auto-radiographic film for detection of hybridizationsignals.

By varying the stringency of the hybridization conditions used toidentify nucleic acids, such as cDNAs or genomic DNAs, which hybridizeto the detectable probe, nucleic acids having different levels ofhomology to the probe can be identified and isolated. Stringency may bevaried by conducting the hybridization at varying temperatures below themelting temperatures of the probes. The melting temperature, Tm, is thetemperature (under defined ionic strength and pH) at which 50% of thetarget sequence hybridizes to a perfectly complementary probe. Verystringent conditions are selected to be equal to or about 5° C. lowerthan the Tm for a particular probe. The melting temperature of the probemay be calculated using the following exemplary formulas. For probesbetween 14 and 70 nucleotides in length the melting temperature (Tm) iscalculated using the formula: Tm=81.5+16.6 (log [Na+])+0.41 (fractionG+C)−(600/N) where N is the length of the probe. If the hybridization iscarried out in a solution containing formamide, the melting temperaturemay be calculated using the equation: Tm=81.5+16.6 (log [Na+])+0.41(fraction G+C)−(0.63% formamide)−(600/N) where N is the length of theprobe. Prehybridization may be carried out in 6×SSC, 5×Denhardt'sreagent, 0.5% SDS, 100 μg denatured fragmented salmon sperm DNA or6×SSC, 5×Denhardt's reagent, 0.5% SDS, 100 μg denatured fragmentedsalmon sperm DNA, 50% formamide. Formulas for SSC and Denhardt's andother solutions are listed, e.g., in Sambrook.

Hybridization is conducted by adding the detectable probe to theprehybridization solutions listed above. Where the probe comprisesdouble stranded DNA, it is denatured before addition to thehybridization solution. The filter is contacted with the hybridizationsolution for a sufficient period of time to allow the probe to hybridizeto cDNAs or genomic DNAs containing sequences complementary thereto orhomologous thereto. For probes over 200 nucleotides in length, thehybridization may be carried out at 15-25° C. below the Tm. For shorterprobes, such as oligonucleotide probes, the hybridization may beconducted at 5-10° C. below the Tm. In one aspect, hybridizations in6×SSC are conducted at approximately 68° C. In one aspect,hybridizations in 50% formamide containing solutions are conducted atapproximately 42° C. All of the foregoing hybridizations would beconsidered to be under conditions of high stringency.

Following hybridization, the filter is washed to remove anynon-specifically bound detectable probe. The stringency used to wash thefilters can also be varied depending on the nature of the nucleic acidsbeing hybridized, the length of the nucleic acids being hybridized, thedegree of complementarity, the nucleotide sequence composition (e.g., GCv. AT content), and the nucleic acid type (e.g., RNA v. DNA). Examplesof progressively higher stringency condition washes are as follows:2×SSC, 0.1% SDS at room temperature for 15 minutes (low stringency);0.1×SSC, 0.5% SDS at room temperature for 30 minutes to 1 hour (moderatestringency); 0.1×SSC, 0.5% SDS for 15 to 30 minutes at between thehybridization temperature and 68° C. (high stringency); and 0.15M NaClfor 15 minutes at 72° C. (very high stringency). A final low stringencywash can be conducted in 0.1×SSC at room temperature. The examples aboveare merely illustrative of one set of conditions that can be used towash filters. One of skill in the art would know that there are numerousrecipes for different stringency washes.

Nucleic acids which have hybridized to the probe can be identified byautoradiography or other conventional techniques. The above proceduremay be modified to identify nucleic acids having decreasing levels ofhomology to the probe sequence. For example, to obtain nucleic acids ofdecreasing homology to the detectable probe, less stringent conditionsmay be used. For example, the hybridization temperature may be decreasedin increments of 5° C. from 68° C. to 42° C. in a hybridization bufferhaving a Na+ concentration of approximately 1M. Following hybridization,the filter may be washed with 2×SSC, 0.5% SDS at the temperature ofhybridization. These conditions are considered to be “moderate”conditions above 50° C. and “low” conditions below 50° C. An example of“moderate” hybridization conditions is when the above hybridization isconducted at 55° C. An example of “low stringency” hybridizationconditions is when the above hybridization is conducted at 45° C.

Alternatively, the hybridization may be carried out in buffers, such as6×SSC, containing formamide at a temperature of 42° C. In this case, theconcentration of formamide in the hybridization buffer may be reduced in5% increments from 50% to 0% to identify clones having decreasing levelsof homology to the probe. Following hybridization, the filter may bewashed with 6×SSC, 0.5% SDS at 50° C. These conditions are considered tobe “moderate” conditions above 25% formamide and “low” conditions below25% formamide. A specific example of “moderate” hybridization conditionsis when the above hybridization is conducted at 30% formamide. Aspecific example of “low stringency” hybridization conditions is whenthe above hybridization is conducted at 10% formamide.

These probes and methods of the invention can be used to isolate nucleicacids having a sequence with at least about 50%, 51%, 52%, 53%, 54%,55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%,69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% homology to a nucleic acid sequence of the inventioncomprising at least about 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150,200, 250, 300, 350, 400, 500, 550, 600, 650, 700, 750, 800, 850, 900,950, 1000, or more consecutive bases thereof, and the sequencescomplementary thereto. Homology may be measured using an alignmentalgorithm, as discussed herein. For example, the homologouspolynucleotides may have a coding sequence which is a naturallyoccurring allelic variant of one of the coding sequences describedherein. Such allelic variants may have a substitution, deletion oraddition of one or more nucleotides when compared to a nucleic acid ofthe invention.

Additionally, the probes and methods of the invention can be used toisolate nucleic acids which encode polypeptides having at least about50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%,64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity (homology) to apolypeptide of the invention comprising at least 5, 10, 15, 20, 25, 30,35, 40, 50, 75, 100, or 150 consecutive amino acids, as determined usinga sequence alignment algorithm (e.g., such as the FASTA version 3.0t78algorithm with the default parameters, or a BLAST 2.2.2 program withexemplary settings as set forth herein).

Inhibiting Expression of Amylase

The invention provides nucleic acids complementary to (e.g., antisensesequences to) the nucleic acid sequences of the invention. Antisensesequences are capable of inhibiting the transport, splicing ortranscription of amylase-encoding genes. The inhibition can be effectedthrough the targeting of genomic DNA or messenger RNA. The transcriptionor function of targeted nucleic acid can be inhibited, for example, byhybridization and/or cleavage. One particularly useful set of inhibitorsprovided by the present invention includes oligonucleotides which areable to either bind amylase gene or message, in either case preventingor inhibiting the production or function of amylase. The association canbe through sequence specific hybridization. Another useful class ofinhibitors includes oligonucleotides which cause inactivation orcleavage of amylase message. The oligonucleotide can have enzymeactivity which causes such cleavage, such as ribozymes. Theoligonucleotide can be chemically modified or conjugated to an enzyme orcomposition capable of cleaving the complementary nucleic acid. A poolof many different such oligonucleotides can be screened for those withthe desired activity.

Antisense Oligonucleotides

The invention provides antisense oligonucleotides capable of bindingamylase message which can inhibit proteolytic activity by targetingmRNA. Strategies for designing antisense oligonucleotides are welldescribed in the scientific and patent literature, and the skilledartisan can design such amylase oligonucleotides using the novelreagents of the invention. For example, gene walking/RNA mappingprotocols to screen for effective antisense oligonucleotides are wellknown in the art, see, e.g., Ho (2000) Methods Enzymol. 314:168-183,describing an RNA mapping assay, which is based on standard moleculartechniques to provide an easy and reliable method for potent antisensesequence selection. See also Smith (2000) Eur. J. Pharm. Sci.11:191-198.

Naturally occurring nucleic acids are used as antisenseoligonucleotides. The antisense oligonucleotides can be of any length;for example, in alternative aspects, the antisense oligonucleotides arebetween about 5 to 100, about 10 to 80, about 15 to 60, about 18 to 40.The optimal length can be determined by routine screening. The antisenseoligonucleotides can be present at any concentration. The optimalconcentration can be determined by routine screening. A wide variety ofsynthetic, non-naturally occurring nucleotide and nucleic acid analoguesare known which can address this potential problem. For example, peptidenucleic acids (PNAs) containing non-ionic backbones, such asN-(2-aminoethyl) glycine units can be used. Antisense oligonucleotideshaving phosphorothioate linkages can also be used, as described in WO97/03211; WO 96/39154; Mata (1997) Toxicol Appl Pharmacol 144:189-197;Antisense Therapeutics, ed. Agrawal (Humana Press, Totowa, N.J., 1996).Antisense oligonucleotides having synthetic DNA backbone analoguesprovided by the invention can also include phosphoro-dithioate,methylphosphonate, phosphoramidate, alkyl phosphotriester, sulfamate,3′-thioacetal, methylene(methylimino), 3′-N-carbamate, and morpholinocarbamate nucleic acids, as described above.

Combinatorial chemistry methodology can be used to create vast numbersof oligonucleotides that can be rapidly screened for specificoligonucleotides that have appropriate binding affinities andspecificities toward any target, such as the sense and antisense amylasesequences of the invention (see, e.g., Gold (1995) J. of Biol. Chem.270:13581-13584).

Inhibitory Ribozymes

The invention provides ribozymes capable of binding amylase message.These ribozymes can inhibit amylase activity by, e.g., targeting mRNA.Strategies for designing ribozymes and selecting the amylase-specificantisense sequence for targeting are well described in the scientificand patent literature, and the skilled artisan can design such ribozymesusing the novel reagents of the invention. Ribozymes act by binding to atarget RNA through the target RNA binding portion of a ribozyme which isheld in close proximity to an enzymatic portion of the RNA that cleavesthe target RNA. Thus, the ribozyme recognizes and binds a target RNAthrough complementary base-pairing, and once bound to the correct site,acts enzymatically to cleave and inactivate the target RNA. Cleavage ofa target RNA in such a manner will destroy its ability to directsynthesis of an encoded protein if the cleavage occurs in the codingsequence. After a ribozyme has bound and cleaved its RNA target, it canbe released from that RNA to bind and cleave new targets repeatedly.

In some circumstances, the enzymatic nature of a ribozyme can beadvantageous over other technologies, such as antisense technology(where a nucleic acid molecule simply binds to a nucleic acid target toblock its transcription, translation or association with anothermolecule) as the effective concentration of ribozyme necessary to effecta therapeutic treatment can be lower than that of an antisenseoligonucleotide. This potential advantage reflects the ability of theribozyme to act enzymatically. Thus, a single ribozyme molecule is ableto cleave many molecules of target RNA. In addition, a ribozyme istypically a highly specific inhibitor, with the specificity ofinhibition depending not only on the base pairing mechanism of binding,but also on the mechanism by which the molecule inhibits the expressionof the RNA to which it binds. That is, the inhibition is caused bycleavage of the RNA target and so specificity is defined as the ratio ofthe rate of cleavage of the targeted RNA over the rate of cleavage ofnon-targeted RNA. This cleavage mechanism is dependent upon factorsadditional to those involved in base pairing. Thus, the specificity ofaction of a ribozyme can be greater than that of antisenseoligonucleotide binding the same RNA site.

The ribozyme of the invention, e.g., an enzymatic ribozyme RNA molecule,can be formed in a hammerhead motif, a hairpin motif, as a hepatitisdelta virus motif, a group I intron motif and/or an RNaseP-like RNA inassociation with an RNA guide sequence. Examples of hammerhead motifsare described by, e.g., Rossi (1992) Aids Research and HumanRetroviruses 8:183; hairpin motifs by Hampel (1989) Biochemistry28:4929, and Hampel (1990) Nuc. Acids Res. 18:299; the hepatitis deltavirus motif by Perrotta (1992) Biochemistry 31:16; the RNaseP motif byGuerrier-Takada (1983) Cell 35:849; and the group I intron by Cech U.S.Pat. No. 4,987,071. The recitation of these specific motifs is notintended to be limiting. Those skilled in the art will recognize that aribozyme of the invention, e.g., an enzymatic RNA molecule of thisinvention, can have a specific substrate binding site complementary toone or more of the target gene RNA regions. A ribozyme of the inventioncan have a nucleotide sequence within or surrounding that substratebinding site which imparts an RNA cleaving activity to the molecule.

RNA interference (RNAi)

In one aspect, the invention provides an RNA inhibitory molecule, aso-called “RNAi” molecule, comprising an amylase sequence of theinvention. The RNAi molecule comprises a double-stranded RNA (dsRNA)molecule. The RNAi can inhibit expression of an amylase gene. In oneaspect, the RNAi is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 ormore duplex nucleotides in length. While the invention is not limited byany particular mechanism of action, the RNAi can enter a cell and causethe degradation of a single-stranded RNA (ssRNA) of similar or identicalsequences, including endogenous mRNAs. When a cell is exposed todouble-stranded RNA (dsRNA), mRNA from the homologous gene isselectively degraded by a process called RNA interference (RNAi). Apossible basic mechanism behind RNAi is the breaking of adouble-stranded RNA (dsRNA) matching a specific gene sequence into shortpieces called short interfering RNA, which trigger the degradation ofmRNA that matches its sequence. In one aspect, the RNAi's of theinvention are used in gene-silencing therapeutics, see, e.g., Shuey(2002) Drug Discov. Today 7:1040-1046. In one aspect, the inventionprovides methods to selectively degrade RNA using the RNAi's of theinvention. The process may be practiced in vitro, ex vivo or in vivo. Inone aspect, the RNAi molecules of the invention can be used to generatea loss-of-function mutation in a cell, an organ or an animal. Methodsfor making and using RNAi molecules for selectively degrade RNA are wellknown in the art, see, e.g., U.S. Pat. Nos. 6,506,559; 6,511,824;6,515,109; 6,489,127.

Modification of Nucleic Acids

The invention provides methods of generating variants of the nucleicacids of the invention, e.g., those encoding an amylase. These methodscan be repeated or used in various combinations to generate amylaseshaving an altered or different activity or an altered or differentstability from that of an amylase encoded by the template nucleic acid.These methods also can be repeated or used in various combinations,e.g., to generate variations in gene/message expression, messagetranslation or message stability. In another aspect, the geneticcomposition of a cell is altered by, e.g., modification of a homologousgene ex vivo, followed by its reinsertion into the cell.

A nucleic acid of the invention can be altered by any means. Forexample, random or stochastic methods, or, non-stochastic, or “directedevolution,” methods, see, e.g., U.S. Pat. No. 6,361,974. Methods forrandom mutation of genes are well known in the art, see, e.g., U.S. Pat.No. 5,830,696. For example, mutagens can be used to randomly mutate agene. Mutagens include, e.g., ultraviolet light or gamma irradiation, ora chemical mutagen, e.g., mitomycin, nitrous acid, photoactivatedpsoralens, alone or in combination, to induce DNA breaks amenable torepair by recombination. Other chemical mutagens include, for example,sodium bisulfate, nitrous acid, hydroxylamine, hydrazine or formic acid.Other mutagens are analogues of nucleotide precursors, e.g.,nitrosoguanidine, 5-bromouracil, 2-aminopurine, or acridine. Theseagents can be added to a PCR reaction in place of the nucleotideprecursor thereby mutating the sequence. Intercalating agents such asproflavine, acriflavine, quinacrine and the like can also be used.

Any technique in molecular biology can be used, e.g., random PCRmutagenesis, see, e.g., Rice (1992) Proc. Natl. Acad. Sci. USA89:5467-5471; or, combinatorial multiple cassette mutagenesis, see,e.g., Crameri (1995) Biotechniques 18:194-196. Alternatively, nucleicacids, e.g., genes, can be reassembled after random, or “stochastic,”fragmentation, see, e.g., U.S. Pat. Nos. 6,291,242; 6,287,862;6,287,861; 5,955,358; 5,830,721; 5,824,514; 5,811,238; 5,605,793. Inalternative aspects, modifications, additions or deletions areintroduced by error-prone PCR, shuffling, oligonucleotide-directedmutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis,cassette mutagenesis, recursive ensemble mutagenesis, exponentialensemble mutagenesis, site-specific mutagenesis, gene reassembly, genesite saturated mutagenesis (GSSM), synthetic ligation reassembly (SLR),recombination, recursive sequence recombination, phosphothioate-modifiedDNA mutagenesis, uracil-containing template mutagenesis, gapped duplexmutagenesis, point mismatch repair mutagenesis, repair-deficient hoststrain mutagenesis, chemical mutagenesis, radiogenic mutagenesis,deletion mutagenesis, restriction-selection mutagenesis,restriction-purification mutagenesis, artificial gene synthesis,ensemble mutagenesis, chimeric nucleic acid multimer creation, and/or acombination of these and other methods.

The following publications describe a variety of recursive recombinationprocedures and/or methods which can be incorporated into the methods ofthe invention: Stemmer (1999) “Molecular breeding of viruses fortargeting and other clinical properties” Tumor Targeting 4:1-4; Ness(1999) Nature Biotechnology 17:893-896; Chang (1999) “Evolution of acytokine using DNA family shuffling” Nature Biotechnology 17:793-797;Minshull (1999) “Protein evolution by molecular breeding” CurrentOpinion in Chemical Biology 3:284-290; Christians (1999) “Directedevolution of thymidine kinase for AZT phosphorylation using DNA familyshuffling” Nature Biotechnology 17:259-264; Crameri (1998) “DNAshuffling of a family of genes from diverse species accelerates directedevolution” Nature 391:288-291; Crameri (1997) “Molecular evolution of anarsenate detoxification pathway by DNA shuffling,” Nature Biotechnology15:436-438; Zhang (1997) “Directed evolution of an effective fucosidasefrom a galactosidase by DNA shuffling and screening” Proc. Natl. Acad.Sci. USA 94:4504-4509; Patten et al. (1997) “Applications of DNAShuffling to Pharmaceuticals and Vaccines” Current Opinion inBiotechnology 8:724-733; Crameri et al. (1996) “Construction andevolution of antibody-phage libraries by DNA shuffling” Nature Medicine2:100-103; Gates et al. (1996) “Affinity selective isolation of ligandsfrom peptide libraries through display on a lac repressor ‘headpiecedimer’” Journal of Molecular Biology 255:373-386; Stemmer (1996) “SexualPCR and Assembly PCR” In: The Encyclopedia of Molecular Biology. VCHPublishers, New York. pp. 447-457; Crameri and Stemmer (1995)“Combinatorial multiple cassette mutagenesis creates all thepermutations of mutant and wildtype cassettes” BioTechniques 18:194-195;Stemmer et al. (1995) “Single-step assembly of a gene and entire plasmidform large numbers of oligodeoxyribonucleotides” Gene, 164:49-53;Stemmer (1995) “The Evolution of Molecular Computation” Science 270:1510; Stemmer (1995) “Searching Sequence Space” Bio/Technology13:549-553; Stemmer (1994) “Rapid evolution of a protein in vitro by DNAshuffling” Nature 370:389-391; and Stemmer (1994) “DNA shuffling byrandom fragmentation and reassembly: In vitro recombination formolecular evolution.” Proc. Natl. Acad. Sci. USA 91:10747-10751.

Mutational methods of generating diversity include, for example,site-directed mutagenesis (Ling et al. (1997) “Approaches to DNAmutagenesis: an overview” Anal Biochem. 254(2): 157-178; Dale et al.(1996) “Oligonucleotide-directed random mutagenesis using thephosphorothioate method” Methods Mol. Biol. 57:369-374; Smith (1985) “Invitro mutagenesis” Ann. Rev. Genet. 19:423-462; Botstein & Shortle(1985) “Strategies and applications of in vitro mutagenesis” Science229:1193-1201; Carter (1986) “Site-directed mutagenesis” Biochem. J.237:1-7; and Kunkel (1987) “The efficiency of oligonucleotide directedmutagenesis” in Nucleic Acids & Molecular Biology (Eckstein, F. andLilley, D. M. J. eds., Springer Verlag, Berlin)); mutagenesis usinguracil containing templates (Kunkel (1985) “Rapid and efficientsite-specific mutagenesis without phenotypic selection” Proc. Natl.Acad. Sci. USA 82:488-492; Kunkel et al. (1987) “Rapid and efficientsite-specific mutagenesis without phenotypic selection” Methods inEnzymol. 154, 367-382; and Bass et al. (1988) “Mutant Trp repressorswith new DNA-binding specificities” Science 242:240-245);oligonucleotide-directed mutagenesis (Methods in Enzymol. 100: 468-500(1983); Methods in Enzymol. 154: 329-350 (1987); Zoller & Smith (1982)“Oligonucleotide-directed mutagenesis using M13-derived vectors: anefficient and general procedure for the production of point mutations inany DNA fragment” Nucleic Acids Res. 10:6487-6500; Zoller & Smith (1983)“Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13vectors” Methods in Enzymol. 100:468-500; and Zoller & Smith (1987)Oligonucleotide-directed mutagenesis: a simple method using twooligonucleotide primers and a single-stranded DNA template” Methods inEnzymol. 154:329-350); phosphorothioate-modified DNA mutagenesis (Tayloret al. (1985) “The use of phosphorothioate-modified DNA in restrictionenzyme reactions to prepare nicked DNA” Nucl. Acids Res. 13: 8749-8764;Taylor et al. (1985) “The rapid generation of oligonucleotide-directedmutations at high frequency using phosphorothioate-modified DNA” Nucl.Acids Res. 13: 8765-8787 (1985); Nakamaye (1986) “Inhibition ofrestriction endonuclease Nci I cleavage by phosphorothioate groups andits application to oligonucleotide-directed mutagenesis” Nucl. AcidsRes. 14: 9679-9698; Sayers et al. (1988) “Y-T Exonucleases inphosphorothioate-based oligonucleotide-directed mutagenesis” Nucl. AcidsRes. 16:791-802; and Sayers et al. (1988) “Strand specific cleavage ofphosphorothioate-containing DNA by reaction with restrictionendonucleases in the presence of ethidium bromide” Nucl. Acids Res. 16:803-814); mutagenesis using gapped duplex DNA (Kramer et al. (1984) “Thegapped duplex DNA approach to oligonucleotide-directed mutationconstruction” Nucl. Acids Res. 12: 9441-9456; Kramer & Fritz (1987)Methods in Enzymol. “Oligonucleotide-directed construction of mutationsvia gapped duplex DNA” 154:350-367; Kramer et al. (1988) “Improvedenzymatic in vitro reactions in the gapped duplex DNA approach tooligonucleotide-directed construction of mutations” Nucl. Acids Res. 16:7207; and Fritz et al. (1988) “Oligonucleotide-directed construction ofmutations: a gapped duplex DNA procedure without enzymatic reactions invitro” Nucl. Acids Res. 16: 6987-6999).

Additional protocols that can be used to practice the invention includepoint mismatch repair (Kramer (1984) “Point Mismatch Repair” Cell38:879-887), mutagenesis using repair-deficient host strains (Carter etal. (1985) “Improved oligonucleotide site-directed mutagenesis using M13vectors” Nucl. Acids Res. 13: 4431-4443; and Carter (1987) “Improvedoligonucleotide-directed mutagenesis using M13 vectors” Methods inEnzymol. 154: 382-403), deletion mutagenesis (Eghtedarzadeh (1986) “Useof oligonucleotides to generate large deletions” Nucl. Acids Res. 14:5115), restriction-selection and restriction-selection andrestriction-purification (Wells et al. (1986) “Importance ofhydrogen-bond formation in stabilizing the transition state ofsubtilisin” Phil. Trans. R. Soc. Lond. A 317: 415-423), mutagenesis bytotal gene synthesis (Nambiar et al. (1984) “Total synthesis and cloningof a gene coding for the ribonuclease S protein” Science 223: 1299-1301;Sakamar and Khorana (1988) “Total synthesis and expression of a gene forthe α-subunit of bovine rod outer segment guanine nucleotide-bindingprotein (transducin)” Nucl. Acids Res. 14: 6361-6372; Wells et al.(1985) “Cassette mutagenesis: an efficient method for generation ofmultiple mutations at defined sites” Gene 34:315-323; and Grundstrom etal. (1985) “Oligonucleotide-directed mutagenesis by microscale‘shot-gun’ gene synthesis” Nucl. Acids Res. 13: 3305-3316),double-strand break repair (Mandecki (1986); Arnold (1993) “Proteinengineering for unusual environments” Current Opinion in Biotechnology4:450-455. “Oligonucleotide-directed double-strand break repair inplasmids of Escherichia coli: a method for site-specific mutagenesis”Proc. Natl. Acad. Sci. USA, 83:7177-7181). Additional details on many ofthe above methods can be found in Methods in Enzymology Volume 154,which also describes useful controls for trouble-shooting problems withvarious mutagenesis methods.

Protocols that can be used to practice the invention are described,e.g., in U.S. Pat. No. 5,605,793 to Stemmer (Feb. 25, 1997), “Methodsfor In Vitro Recombination;” U.S. Pat. No. 5,811,238 to Stemmer et al.(Sep. 22, 1998) “Methods for Generating Polynucleotides having DesiredCharacteristics by Iterative Selection and Recombination;” U.S. Pat. No.5,830,721 to Stemmer et al. (Nov. 3, 1998), “DNA Mutagenesis by RandomFragmentation and Reassembly;” U.S. Pat. No. 5,834,252 to Stemmer, etal. (Nov. 10, 1998) “End-Complementary Polymerase Reaction;” U.S. Pat.No. 5,837,458 to Minshull, et al. (Nov. 17, 1998), “Methods andCompositions for Cellular and Metabolic Engineering;” WO 95/22625,Stemmer and Crameri, “Mutagenesis by Random Fragmentation andReassembly;” WO 96/33207 by Stemmer and Lipschutz “End ComplementaryPolymerase Chain Reaction;” WO 97/20078 by Stemmer and Crameri “Methodsfor Generating Polynucleotides having Desired Characteristics byIterative Selection and Recombination;” WO 97/35966 by Minshull andStemmer, “Methods and Compositions for Cellular and MetabolicEngineering;” WO 99/41402 by Punnonen et al. “Targeting of GeneticVaccine Vectors;” WO 99/41383 by Punnonen et al. “Antigen LibraryImmunization;” WO 99/41369 by Punnonen et al. “Genetic Vaccine VectorEngineering;” WO 99/41368 by Punnonen et al. “Optimization ofImmunomodulatory Properties of Genetic Vaccines;” EP 752008 by Stemmerand Crameri, “DNA Mutagenesis by Random Fragmentation and Reassembly;”EP 0932670 by Stemmer “Evolving Cellular DNA Uptake by RecursiveSequence Recombination;” WO 99/23107 by Stemmer et al., “Modification ofVirus Tropism and Host Range by Viral Genome Shuffling;” WO 99/21979 byApt et al., “Human Papillomavirus Vectors;” WO 98/31837 by del Cardayreet al. “Evolution of Whole Cells and Organisms by Recursive SequenceRecombination;” WO 98/27230 by Patten and Stemmer, “Methods andCompositions for Polypeptide Engineering;” WO 98/27230 by Stemmer etal., “Methods for Optimization of Gene Therapy by Recursive SequenceShuffling and Selection,” WO 00/00632, “Methods for Generating HighlyDiverse Libraries,” WO 00/09679, “Methods for Obtaining in VitroRecombined Polynucleotide Sequence Banks and Resulting Sequences,” WO98/42832 by Arnold et al., “Recombination of Polynucleotide SequencesUsing Random or Defined Primers,” WO 99/29902 by Arnold et al., “Methodfor Creating Polynucleotide and Polypeptide Sequences,” WO 98/41653 byVind, “An in Vitro Method for Construction of a DNA Library,” WO98/41622 by Borchert et al., “Method for Constructing a Library UsingDNA Shuffling,” and WO 98/42727 by Pati and Zarling, “SequenceAlterations using Homologous Recombination.”

Protocols that can be used to practice the invention (providing detailsregarding various diversity generating methods) are described, e.g., inU.S. patent application Ser. No. 09/407,800, “SHUFFLING OF CODON ALTEREDGENES” by Patten et al. filed Sep. 28, 1999; “EVOLUTION OF WHOLE CELLSAND ORGANISMS BY RECURSIVE SEQUENCE RECOMBINATION” by del Cardayre etal., U.S. Pat. No. 6,379,964; “OLIGONUCLEOTIDE MEDIATED NUCLEIC ACIDRECOMBINATION” by Crameri et al., U.S. Pat. Nos. 6,319,714; 6,368,861;6,376,246; 6,423,542; 6,426,224 and PCT/US00/01203; “USE OF CODON-VARIEDOLIGONUCLEOTIDE SYNTHESIS FOR SYNTHETIC SHUFFLING” by Welch et al., U.S.Pat. No. 6,436,675; “METHODS FOR MAKING CHARACTER STRINGS,POLYNUCLEOTIDES & POLYPEPTIDES HAVING DESIRED CHARACTERISTICS” bySelifonov et al., filed Jan. 18, 2000, (PCT/US00/01202) and, e.g.“METHODS FOR MAKING CHARACTER STRINGS, POLYNUCLEOTIDES & POLYPEPTIDESHAVING DESIRED CHARACTERISTICS” by Selifonov et al., filed Jul. 18, 2000(U.S. Ser. No. 09/618,579); “METHODS OF POPULATING DATA STRUCTURES FORUSE IN EVOLUTIONARY SIMULATIONS” by Selifonov and Stemmer, filed Jan.18, 2000 (PCT/US00/01138); and “SINGLE-STRANDED NUCLEIC ACIDTEMPLATE-MEDIATED RECOMBINATION AND NUCLEIC ACID FRAGMENT ISOLATION” byAffholter, filed Sep. 6, 2000 (U.S. Ser. No. 09/656,549); and U.S. Pat.Nos. 6,177,263; 6,153,410.

Non-stochastic, or “directed evolution,” methods include, e.g.,saturation mutagenesis (GSSM), synthetic ligation reassembly (SLR), or acombination thereof are used to modify the nucleic acids of theinvention to generate amylases with new or altered properties (e.g.,activity under highly acidic or alkaline conditions, high temperatures,and the like). Polypeptides encoded by the modified nucleic acids can bescreened for an activity before testing for proteolytic or otheractivity. Any testing modality or protocol can be used, e.g., using acapillary array platform. See, e.g., U.S. Pat. Nos. 6,361,974;6,280,926; 5,939,250.

Saturation Mutagenesis, or, GSSM

In one aspect, codon primers containing a degenerate N,N,G/T sequenceare used to introduce point mutations into a polynucleotide, e.g., anamylase or an antibody of the invention, so as to generate a set ofprogeny polypeptides in which a full range of single amino acidsubstitutions is represented at each amino acid position, e.g., an aminoacid residue in an enzyme active site or ligand binding site targeted tobe modified. These oligonucleotides can comprise a contiguous firsthomologous sequence, a degenerate N,N,G/T sequence, and, optionally, asecond homologous sequence. The downstream progeny translationalproducts from the use of such oligonucleotides include all possibleamino acid changes at each amino acid site along the polypeptide,because the degeneracy of the N,N,G/T sequence includes codons for all20 amino acids. In one aspect, one such degenerate oligonucleotide(comprised of, e.g., one degenerate N,N,G/T cassette) is used forsubjecting each original codon in a parental polynucleotide template toa full range of codon substitutions. In another aspect, at least twodegenerate cassettes are used—either in the same oligonucleotide or not,for subjecting at least two original codons in a parental polynucleotidetemplate to a full range of codon substitutions. For example, more thanone N,N,G/T sequence can be contained in one oligonucleotide tointroduce amino acid mutations at more than one site. This plurality ofN,N,G/T sequences can be directly contiguous, or separated by one ormore additional nucleotide sequence(s). In another aspect,oligonucleotides serviceable for introducing additions and deletions canbe used either alone or in combination with the codons containing anN,N,G/T sequence, to introduce any combination or permutation of aminoacid additions, deletions, and/or substitutions.

In one aspect, simultaneous mutagenesis of two or more contiguous aminoacid positions is done using an oligonucleotide that contains contiguousN,N,G/T triplets, i.e. a degenerate (N,N,G/T)n sequence. In anotheraspect, degenerate cassettes having less degeneracy than the N,N,G/Tsequence are used. For example, it may be desirable in some instances touse (e.g. in an oligonucleotide) a degenerate triplet sequence comprisedof only one N, where said N can be in the first second or third positionof the triplet. Any other bases including any combinations andpermutations thereof can be used in the remaining two positions of thetriplet. Alternatively, it may be desirable in some instances to use(e.g. in an oligo) a degenerate N,N,N triplet sequence.

In one aspect, use of degenerate triplets (e.g., N,N,G/T triplets)allows for systematic and easy generation of a full range of possiblenatural amino acids (for a total of 20 amino acids) into each and everyamino acid position in a polypeptide (in alternative aspects, themethods also include generation of less than all possible substitutionsper amino acid residue, or codon, position). For example, for a 100amino acid polypeptide, 2000 distinct species (i.e. 20 possible aminoacids per position×100 amino acid positions) can be generated. Throughthe use of an oligonucleotide or set of oligonucleotides containing adegenerate N,N,G/T triplet, 32 individual sequences can code for all 20possible natural amino acids. Thus, in a reaction vessel in which aparental polynucleotide sequence is subjected to saturation mutagenesisusing at least one such oligonucleotide, there are generated 32 distinctprogeny polynucleotides encoding 20 distinct polypeptides. In contrast,the use of a non-degenerate oligonucleotide in site-directed mutagenesisleads to only one progeny polypeptide product per reaction vessel.Nondegenerate oligonucleotides can optionally be used in combinationwith degenerate primers disclosed; for example, nondegenerateoligonucleotides can be used to generate specific point mutations in aworking polynucleotide. This provides one means to generate specificsilent point mutations, point mutations leading to corresponding aminoacid changes, and point mutations that cause the generation of stopcodons and the corresponding expression of polypeptide fragments.

In one aspect, each saturation mutagenesis reaction vessel containspolynucleotides encoding at least 20 progeny polypeptide (e.g.,amylases) molecules such that all 20 natural amino acids are representedat the one specific amino acid position corresponding to the codonposition mutagenized in the parental polynucleotide (other aspects useless than all 20 natural combinations). The 32-fold degenerate progenypolypeptides generated from each saturation mutagenesis reaction vesselcan be subjected to clonal amplification (e.g. cloned into a suitablehost, e.g., E. coli host, using, e.g., an expression vector) andsubjected to expression screening. When an individual progenypolypeptide is identified by screening to display a favorable change inproperty (when compared to the parental polypeptide, such as increasedproteolytic activity under alkaline or acidic conditions), it can besequenced to identify the correspondingly favorable amino acidsubstitution contained therein.

In one aspect, upon mutagenizing each and every amino acid position in aparental polypeptide using saturation mutagenesis as disclosed herein,favorable amino acid changes may be identified at more than one aminoacid position. One or more new progeny molecules can be generated thatcontain a combination of all or part of these favorable amino acidsubstitutions. For example, if 2 specific favorable amino acid changesare identified in each of 3 amino acid positions in a polypeptide, thepermutations include 3 possibilities at each position (no change fromthe original amino acid, and each of two favorable changes) and 3positions. Thus, there are 3×3×3 or 27 total possibilities, including 7that were previously examined—6 single point mutations (i.e. 2 at eachof three positions) and no change at any position.

In another aspect, site-saturation mutagenesis can be used together withanother stochastic or non-stochastic means to vary sequence, e.g.,synthetic ligation reassembly (see below), shuffling, chimerization,recombination and other mutagenizing processes and mutagenizing agents.This invention provides for the use of any mutagenizing process(es),including saturation mutagenesis, in an iterative manner.

Synthetic Ligation Reassembly (SLR)

The invention provides a non-stochastic gene modification system termed“synthetic ligation reassembly,” or simply “SLR,” a “directed evolutionprocess,” to generate polypeptides, e.g., amylases or antibodies of theinvention, with new or altered properties. SLR is a method of ligatingoligonucleotide fragments together non-stochastically. This methoddiffers from stochastic oligonucleotide shuffling in that the nucleicacid building blocks are not shuffled, concatenated or chimerizedrandomly, but rather are assembled non-stochastically. See, e.g., U.S.patent application Ser. No. 09/332,835 entitled “Synthetic LigationReassembly in Directed Evolution” and filed on Jun. 14, 1999 (“U.S. Ser.No. 09/332,835”). In one aspect, SLR comprises the following steps: (a)providing a template polynucleotide, wherein the template polynucleotidecomprises sequence encoding a homologous gene; (b) providing a pluralityof building block polynucleotides, wherein the building blockpolynucleotides are designed to cross-over reassemble with the templatepolynucleotide at a predetermined sequence, and a building blockpolynucleotide comprises a sequence that is a variant of the homologousgene and a sequence homologous to the template polynucleotide flankingthe variant sequence; (c) combining a building block polynucleotide witha template polynucleotide such that the building block polynucleotidecross-over reassembles with the template polynucleotide to generatepolynucleotides comprising homologous gene sequence variations.

SLR does not depend on the presence of high levels of homology betweenpolynucleotides to be rearranged. Thus, this method can be used tonon-stochastically generate libraries (or sets) of progeny moleculescomprised of over 10100 different chimeras. SLR can be used to generatelibraries comprised of over 101000 different progeny chimeras. Thus,aspects of the present invention include non-stochastic methods ofproducing a set of finalized chimeric nucleic acid molecule shaving anoverall assembly order that is chosen by design. This method includesthe steps of generating by design a plurality of specific nucleic acidbuilding blocks having serviceable mutually compatible ligatable ends,and assembling these nucleic acid building blocks, such that a designedoverall assembly order is achieved.

The mutually compatible ligatable ends of the nucleic acid buildingblocks to be assembled are considered to be “serviceable” for this typeof ordered assembly if they enable the building blocks to be coupled inpredetermined orders. Thus, the overall assembly order in which thenucleic acid building blocks can be coupled is specified by the designof the ligatable ends. If more than one assembly step is to be used,then the overall assembly order in which the nucleic acid buildingblocks can be coupled is also specified by the sequential order of theassembly step(s). In one aspect, the annealed building pieces aretreated with an enzyme, such as a ligase (e.g. T4 DNA ligase), toachieve covalent bonding of the building pieces.

In one aspect, the design of the oligonucleotide building blocks isobtained by analyzing a set of progenitor nucleic acid sequencetemplates that serve as a basis for producing a progeny set of finalizedchimeric polynucleotides. These parental oligonucleotide templates thusserve as a source of sequence information that aids in the design of thenucleic acid building blocks that are to be mutagenized, e.g.,chimerized or shuffled. In one aspect of this method, the sequences of aplurality of parental nucleic acid templates are aligned in order toselect one or more demarcation points. The demarcation points can belocated at an area of homology, and are comprised of one or morenucleotides. These demarcation points are preferably shared by at leasttwo of the progenitor templates. The demarcation points can thereby beused to delineate the boundaries of oligonucleotide building blocks tobe generated in order to rearrange the parental polynucleotides. Thedemarcation points identified and selected in the progenitor moleculesserve as potential chimerization points in the assembly of the finalchimeric progeny molecules. A demarcation point can be an area ofhomology (comprised of at least one homologous nucleotide base) sharedby at least two parental polynucleotide sequences. Alternatively, ademarcation point can be an area of homology that is shared by at leasthalf of the parental polynucleotide sequences, or, it can be an area ofhomology that is shared by at least two thirds of the parentalpolynucleotide sequences. Even more preferably a serviceable demarcationpoints is an area of homology that is shared by at least three fourthsof the parental polynucleotide sequences, or, it can be shared by atalmost all of the parental polynucleotide sequences. In one aspect, ademarcation point is an area of homology that is shared by all of theparental polynucleotide sequences.

In one aspect, a ligation reassembly process is performed exhaustivelyin order to generate an exhaustive library of progeny chimericpolynucleotides. In other words, all possible ordered combinations ofthe nucleic acid building blocks are represented in the set of finalizedchimeric nucleic acid molecules. At the same time, in another aspect,the assembly order (i.e. the order of assembly of each building block inthe 5′ to 3 sequence of each finalized chimeric nucleic acid) in eachcombination is by design (or non-stochastic) as described above. Becauseof the non-stochastic nature of this invention, the possibility ofunwanted side products is greatly reduced.

In another aspect, the ligation reassembly method is performedsystematically. For example, the method is performed in order togenerate a systematically compartmentalized library of progenymolecules, with compartments that can be screened systematically, e.g.one by one. In other words this invention provides that, through theselective and judicious use of specific nucleic acid building blocks,coupled with the selective and judicious use of sequentially steppedassembly reactions, a design can be achieved where specific sets ofprogeny products are made in each of several reaction vessels. Thisallows a systematic examination and screening procedure to be performed.Thus, these methods allow a potentially very large number of progenymolecules to be examined systematically in smaller groups. Because ofits ability to perform chimerizations in a manner that is highlyflexible yet exhaustive and systematic as well, particularly when thereis a low level of homology among the progenitor molecules, these methodsprovide for the generation of a library (or set) comprised of a largenumber of progeny molecules. Because of the non-stochastic nature of theinstant ligation reassembly invention, the progeny molecules generatedpreferably comprise a library of finalized chimeric nucleic acidmolecules having an overall assembly order that is chosen by design. Thesaturation mutagenesis and optimized directed evolution methods also canbe used to generate different progeny molecular species. It isappreciated that the invention provides freedom of choice and controlregarding the selection of demarcation points, the size and number ofthe nucleic acid building blocks, and the size and design of thecouplings. It is appreciated, furthermore, that the requirement forintermolecular homology is highly relaxed for the operability of thisinvention. In fact, demarcation points can even be chosen in areas oflittle or no intermolecular homology. For example, because of codonwobble, i.e. the degeneracy of codons, nucleotide substitutions can beintroduced into nucleic acid building blocks without altering the aminoacid originally encoded in the corresponding progenitor template.Alternatively, a codon can be altered such that the coding for anoriginally amino acid is altered. This invention provides that suchsubstitutions can be introduced into the nucleic acid building block inorder to increase the incidence of intermolecular homologous demarcationpoints and thus to allow an increased number of couplings to be achievedamong the building blocks, which in turn allows a greater number ofprogeny chimeric molecules to be generated.

In another aspect, the synthetic nature of the step in which thebuilding blocks are generated allows the design and introduction ofnucleotides (e.g., one or more nucleotides, which may be, for example,codons or introns or regulatory sequences) that can later be optionallyremoved in an in vitro process (e.g. by mutagenesis) or in an in vivoprocess (e.g. by utilizing the gene splicing ability of a hostorganism). It is appreciated that in many instances the introduction ofthese nucleotides may also be desirable for many other reasons inaddition to the potential benefit of creating a serviceable demarcationpoint.

In one aspect, a nucleic acid building block is used to introduce anintron. Thus, functional introns are introduced into a man-made genemanufactured according to the methods described herein. The artificiallyintroduced intron(s) can be functional in a host cells for gene splicingmuch in the way that naturally-occurring introns serve functionally ingene splicing.

Optimized Directed Evolution System

The invention provides a non-stochastic gene modification system termed“optimized directed evolution system” to generate polypeptides, e.g.,amylases or antibodies of the invention, with new or altered properties.Optimized directed evolution is directed to the use of repeated cyclesof reductive reassortment, recombination and selection that allow forthe directed molecular evolution of nucleic acids through recombination.Optimized directed evolution allows generation of a large population ofevolved chimeric sequences, wherein the generated population issignificantly enriched for sequences that have a predetermined number ofcrossover events.

A crossover event is a point in a chimeric sequence where a shift insequence occurs from one parental variant to another parental variant.Such a point is normally at the juncture of where oligonucleotides fromtwo parents are ligated together to form a single sequence. This methodallows calculation of the correct concentrations of oligonucleotidesequences so that the final chimeric population of sequences is enrichedfor the chosen number of crossover events. This provides more controlover choosing chimeric variants having a predetermined number ofcrossover events.

In addition, this method provides a convenient means for exploring atremendous amount of the possible protein variant space in comparison toother systems. Previously, if one generated, for example, 10¹³ chimericmolecules during a reaction, it would be extremely difficult to testsuch a high number of chimeric variants for a particular activity.Moreover, a significant portion of the progeny population would have avery high number of crossover events which resulted in proteins thatwere less likely to have increased levels of a particular activity. Byusing these methods, the population of chimerics molecules can beenriched for those variants that have a particular number of crossoverevents. Thus, although one can still generate 10¹³ chimeric moleculesduring a reaction, each of the molecules chosen for further analysismost likely has, for example, only three crossover events. Because theresulting progeny population can be skewed to have a predeterminednumber of crossover events, the boundaries on the functional varietybetween the chimeric molecules is reduced. This provides a moremanageable number of variables when calculating which oligonucleotidefrom the original parental polynucleotides might be responsible foraffecting a particular trait.

One method for creating a chimeric progeny polynucleotide sequence is tocreate oligonucleotides corresponding to fragments or portions of eachparental sequence. Each oligonucleotide preferably includes a uniqueregion of overlap so that mixing the oligonucleotides together resultsin a new variant that has each oligonucleotide fragment assembled in thecorrect order. Additional information can also be found, e.g., in U.S.Ser. No. 09/332,835; U.S. Pat. No. 6,361,974.

The number of oligonucleotides generated for each parental variant bearsa relationship to the total number of resulting crossovers in thechimeric molecule that is ultimately created. For example, threeparental nucleotide sequence variants might be provided to undergo aligation reaction in order to find a chimeric variant having, forexample, greater activity at high temperature. As one example, a set of50 oligonucleotide sequences can be generated corresponding to eachportions of each parental variant. Accordingly, during the ligationreassembly process there could be up to 50 crossover events within eachof the chimeric sequences. The probability that each of the generatedchimeric polynucleotides will contain oligonucleotides from eachparental variant in alternating order is very low. If eacholigonucleotide fragment is present in the ligation reaction in the samemolar quantity it is likely that in some positions oligonucleotides fromthe same parental polynucleotide will ligate next to one another andthus not result in a crossover event. If the concentration of eacholigonucleotide from each parent is kept constant during any ligationstep in this example, there is a ⅓ chance (assuming 3 parents) that anoligonucleotide from the same parental variant will ligate within thechimeric sequence and produce no crossover.

Accordingly, a probability density function (PDF) can be determined topredict the population of crossover events that are likely to occurduring each step in a ligation reaction given a set number of parentalvariants, a number of oligonucleotides corresponding to each variant,and the concentrations of each variant during each step in the ligationreaction. The statistics and mathematics behind determining the PDF isdescribed below. By utilizing these methods, one can calculate such aprobability density function, and thus enrich the chimeric progenypopulation for a predetermined number of crossover events resulting froma particular ligation reaction. Moreover, a target number of crossoverevents can be predetermined, and the system then programmed to calculatethe starting quantities of each parental oligonucleotide during eachstep in the ligation reaction to result in a probability densityfunction that centers on the predetermined number of crossover events.These methods are directed to the use of repeated cycles of reductivereassortment, recombination and selection that allow for the directedmolecular evolution of a nucleic acid encoding a polypeptide throughrecombination. This system allows generation of a large population ofevolved chimeric sequences, wherein the generated population issignificantly enriched for sequences that have a predetermined number ofcrossover events. A crossover event is a point in a chimeric sequencewhere a shift in sequence occurs from one parental variant to anotherparental variant. Such a point is normally at the juncture of whereoligonucleotides from two parents are ligated together to form a singlesequence. The method allows calculation of the correct concentrations ofoligonucleotide sequences so that the final chimeric population ofsequences is enriched for the chosen number of crossover events. Thisprovides more control over choosing chimeric variants having apredetermined number of crossover events.

In addition, these methods provide a convenient means for exploring atremendous amount of the possible protein variant space in comparison toother systems. By using the methods described herein, the population ofchimerics molecules can be enriched for those variants that have aparticular number of crossover events. Thus, although one can stillgenerate 10¹³ chimeric molecules during a reaction, each of themolecules chosen for further analysis most likely has, for example, onlythree crossover events. Because the resulting progeny population can beskewed to have a predetermined number of crossover events, theboundaries on the functional variety between the chimeric molecules isreduced. This provides a more manageable number of variables whencalculating which oligonucleotide from the original parentalpolynucleotides might be responsible for affecting a particular trait.

In one aspect, the method creates a chimeric progeny polynucleotidesequence by creating oligonucleotides corresponding to fragments orportions of each parental sequence. Each oligonucleotide preferablyincludes a unique region of overlap so that mixing the oligonucleotidestogether results in a new variant that has each oligonucleotide fragmentassembled in the correct order. See also U.S. Ser. No. 09/332,835.

Determining Crossover Events

Aspects of the invention include a system and software that receive adesired crossover probability density function (PDF), the number ofparent genes to be reassembled, and the number of fragments in thereassembly as inputs. The output of this program is a “fragment PDF”that can be used to determine a recipe for producing reassembled genes,and the estimated crossover PDF of those genes. The processing describedherein is preferably performed in MATLAB™ (The Mathworks, Natick, Mass.)a programming language and development environment for technicalcomputing.

Iterative Processes

In practicing the invention, these processes can be iterativelyrepeated. For example, a nucleic acid (or, the nucleic acid) responsiblefor an altered or new amylase phenotype is identified, re-isolated,again modified, re-tested for activity. This process can be iterativelyrepeated until a desired phenotype is engineered. For example, an entirebiochemical anabolic or catabolic pathway can be engineered into a cell,including, e.g., starch hydrolysis activity.

Similarly, if it is determined that a particular oligonucleotide has noaffect at all on the desired trait (e.g., a new amylase phenotype), itcan be removed as a variable by synthesizing larger parentaloligonucleotides that include the sequence to be removed. Sinceincorporating the sequence within a larger sequence prevents anycrossover events, there will no longer be any variation of this sequencein the progeny polynucleotides. This iterative practice of determiningwhich oligonucleotides are most related to the desired trait, and whichare unrelated, allows more efficient exploration all of the possibleprotein variants that might be provide a particular trait or activity.

In Vivo Shuffling

In vivo shuffling of molecules is use in methods of the invention thatprovide variants of polypeptides of the invention, e.g., antibodies,amylases, and the like. In vivo shuffling can be performed utilizing thenatural property of cells to recombine multimers. While recombination invivo has provided the major natural route to molecular diversity,genetic recombination remains a relatively complex process thatinvolves 1) the recognition of homologies; 2) strand cleavage, strandinvasion, and metabolic steps leading to the production of recombinantchiasma; and finally 3) the resolution of chiasma into discreterecombined molecules. The formation of the chiasma requires therecognition of homologous sequences.

In one aspect, the invention provides a method for producing a hybridpolynucleotide from at least a first polynucleotide (e.g., an amylase ofthe invention) and a second polynucleotide (e.g., an enzyme, such as anamylase of the invention or any other amylase, or, a tag or an epitope).The invention can be used to produce a hybrid polynucleotide byintroducing at least a first polynucleotide and a second polynucleotidewhich share at least one region of partial sequence homology into asuitable host cell. The regions of partial sequence homology promoteprocesses which result in sequence reorganization producing a hybridpolynucleotide. The term “hybrid polynucleotide”, as used herein, is anynucleotide sequence which results from the method of the presentinvention and contains sequence from at least two originalpolynucleotide sequences. Such hybrid polynucleotides can result fromintermolecular recombination events which promote sequence integrationbetween DNA molecules. In addition, such hybrid polynucleotides canresult from intramolecular reductive reassortment processes whichutilize repeated sequences to alter a nucleotide sequence within a DNAmolecule.

Producing Sequence Variants

The invention also provides additional methods for making sequencevariants of the nucleic acid (e.g., amylase) sequences of the invention.The invention also provides additional methods for isolating amylasesusing the nucleic acids and polypeptides of the invention. In oneaspect, the invention provides for variants of an amylase codingsequence (e.g., a gene, cDNA or message) of the invention, which can bealtered by any means, including, e.g., random or stochastic methods, or,non-stochastic, or “directed evolution,” methods, as described above.

The isolated variants may be naturally occurring. Variant can also becreated in vitro. Variants may be created using genetic engineeringtechniques such as site directed mutagenesis, random chemicalmutagenesis, Exonuclease III deletion procedures, and standard cloningtechniques. Alternatively, such variants, fragments, analogs, orderivatives may be created using chemical synthesis or modificationprocedures. Other methods of making variants are also familiar to thoseskilled in the art. These include procedures in which nucleic acidsequences obtained from natural isolates are modified to generatenucleic acids which encode polypeptides having characteristics whichenhance their value in industrial or laboratory applications. In suchprocedures, a large number of variant sequences having one or morenucleotide differences with respect to the sequence obtained from thenatural isolate are generated and characterized. These nucleotidedifferences can result in amino acid changes with respect to thepolypeptides encoded by the nucleic acids from the natural isolates.

For example, variants may be created using error prone PCR. In errorprone PCR, PCR is performed under conditions where the copying fidelityof the DNA polymerase is low, such that a high rate of point mutationsis obtained along the entire length of the PCR product. Error prone PCRis described, e.g., in Leung, D. W., et al., Technique, 1:11-15, 1989)and Caldwell, R. C. & Joyce G. F., PCR Methods Applic., 2:28-33, 1992.Briefly, in such procedures, nucleic acids to be mutagenized are mixedwith PCR primers, reaction buffer, MgCl₂, MnCl₂, Taq polymerase and anappropriate concentration of dNTPs for achieving a high rate of pointmutation along the entire length of the PCR product. For example, thereaction may be performed using 20 fmoles of nucleic acid to bemutagenized, 30 pmole of each PCR primer, a reaction buffer comprising50 mM KCl, 10 mM Tris HCl (pH 8.3) and 0.01% gelatin, 7 mM MgCl2, 0.5 mMMnCl₂, 5 units of Taq polymerase, 0.2 mM dGTP, 0.2 mM dATP, 1 mM dCTP,and 1 mM dTTP. PCR may be performed for 30 cycles of 94° C. for 1 min,45° C. for 1 min, and 72° C. for 1 min. However, it will be appreciatedthat these parameters may be varied as appropriate. The mutagenizednucleic acids are cloned into an appropriate vector and the activitiesof the polypeptides encoded by the mutagenized nucleic acids isevaluated.

Variants may also be created using oligonucleotide directed mutagenesisto generate site-specific mutations in any cloned DNA of interest.Oligonucleotide mutagenesis is described, e.g., in Reidhaar-Olson (1988)Science 241:53-57. Briefly, in such procedures a plurality of doublestranded oligonucleotides bearing one or more mutations to be introducedinto the cloned DNA are synthesized and inserted into the cloned DNA tobe mutagenized. Clones containing the mutagenized DNA are recovered andthe activities of the polypeptides they encode are assessed.

Another method for generating variants is assembly PCR. Assembly PCRinvolves the assembly of a PCR product from a mixture of small DNAfragments. A large number of different PCR reactions occur in parallelin the same vial, with the products of one reaction priming the productsof another reaction. Assembly PCR is described in, e.g., U.S. Pat. No.5,965,408.

Still another method of generating variants is sexual PCR mutagenesis.In sexual PCR mutagenesis, forced homologous recombination occursbetween DNA molecules of different but highly related DNA sequence invitro, as a result of random fragmentation of the DNA molecule based onsequence homology, followed by fixation of the crossover by primerextension in a PCR reaction. Sexual PCR mutagenesis is described, e.g.,in Stemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751. Briefly, insuch procedures a plurality of nucleic acids to be recombined aredigested with DNase to generate fragments having an average size of50-200 nucleotides. Fragments of the desired average size are purifiedand resuspended in a PCR mixture. PCR is conducted under conditionswhich facilitate recombination between the nucleic acid fragments. Forexample, PCR may be performed by resuspending the purified fragments ata concentration of 10-30 ng/:1 in a solution of 0.2 mM of each dNTP, 2.2mM MgCl2, 50 mM KCL, 10 mM Tris HCl, pH 9.0, and 0.1% Triton X-100. 2.5units of Taq polymerase per 100:1 of reaction mixture is added and PCRis performed using the following regime: 94° C. for 60 seconds, 94° C.for 30 seconds, 50-55° C. for 30 seconds, 72° C. for 30 seconds (30-45times) and 72° C. for 5 minutes. However, it will be appreciated thatthese parameters may be varied as appropriate. In some aspects,oligonucleotides may be included in the PCR reactions. In other aspects,the Klenow fragment of DNA polymerase I may be used in a first set ofPCR reactions and Taq polymerase may be used in a subsequent set of PCRreactions. Recombinant sequences are isolated and the activities of thepolypeptides they encode are assessed.

Variants may also be created by in vivo mutagenesis. In some aspects,random mutations in a sequence of interest are generated by propagatingthe sequence of interest in a bacterial strain, such as an E. colistrain, which carries mutations in one or more of the DNA repairpathways. Such “mutator” strains have a higher random mutation rate thanthat of a wild-type parent. Propagating the DNA in one of these strainswill eventually generate random mutations within the DNA. Mutatorstrains suitable for use for in vivo mutagenesis are described, e.g., inPCT Publication No. WO 91/16427.

Variants may also be generated using cassette mutagenesis. In cassettemutagenesis a small region of a double stranded DNA molecule is replacedwith a synthetic oligonucleotide “cassette” that differs from the nativesequence. The oligonucleotide often contains completely and/or partiallyrandomized native sequence.

Recursive ensemble mutagenesis may also be used to generate variants.Recursive ensemble mutagenesis is an algorithm for protein engineering(protein mutagenesis) developed to produce diverse populations ofphenotypically related mutants whose members differ in amino acidsequence. This method uses a feedback mechanism to control successiverounds of combinatorial cassette mutagenesis. Recursive ensemblemutagenesis is described, e.g., in Arkin (1992) Proc. Natl. Acad. Sci.USA 89:7811-7815.

In some aspects, variants are created using exponential ensemblemutagenesis. Exponential ensemble mutagenesis is a process forgenerating combinatorial libraries with a high percentage of unique andfunctional mutants, wherein small groups of residues are randomized inparallel to identify, at each altered position, amino acids which leadto functional proteins. Exponential ensemble mutagenesis is described,e.g., in Delegrave (1993) Biotechnology Res. 11:1548-1552. Random andsite-directed mutagenesis are described, e.g., in Arnold (1993) CurrentOpinion in Biotechnology 4:450-455.

In some aspects, the variants are created using shuffling procedureswherein portions of a plurality of nucleic acids which encode distinctpolypeptides are fused together to create chimeric nucleic acidsequences which encode chimeric polypeptides as described in, e.g., U.S.Pat. Nos. 5,965,408; 5,939,250 (see also discussion, above).

The invention also provides variants of polypeptides of the invention(e.g., amylases) comprising sequences in which one or more of the aminoacid residues (e.g., of an exemplary polypeptide of the invention) aresubstituted with a conserved or non-conserved amino acid residue (e.g.,a conserved amino acid residue) and such substituted amino acid residuemay or may not be one encoded by the genetic code. Conservativesubstitutions are those that substitute a given amino acid in apolypeptide by another amino acid of like characteristics. Thus,polypeptides of the invention include those with conservativesubstitutions of sequences of the invention, e.g., the exemplarypolypeptides of the invention, including but not limited to thefollowing replacements: replacements of an aliphatic amino acid such asAlanine, Valine, Leucine and Isoleucine with another aliphatic aminoacid; replacement of a Serine with a Threonine or vice versa;replacement of an acidic residue such as Aspartic acid and Glutamic acidwith another acidic residue; replacement of a residue bearing an amidegroup, such as Asparagine and Glutamine, with another residue bearing anamide group; exchange of a basic residue such as Lysine and Argininewith another basic residue; and replacement of an aromatic residue suchas Phenylalanine, Tyrosine with another aromatic residue. Other variantsare those in which one or more of the amino acid residues of thepolypeptides of the invention includes a substituent group.

Other variants within the scope of the invention are those in which thepolypeptide is associated with another compound, such as a compound toincrease the half-life of the polypeptide, for example, polyethyleneglycol.

Additional variants within the scope of the invention are those in whichadditional amino acids are fused to the polypeptide, such as a leadersequence, a secretory sequence, a proprotein sequence or a sequencewhich facilitates purification, enrichment, or stabilization of thepolypeptide.

In some aspects, the variants, fragments, derivatives and analogs of thepolypeptides of the invention retain the same biological function oractivity as the exemplary polypeptides, e.g., amylase activity, asdescribed herein. In other aspects, the variant, fragment, derivative,or analog includes a proprotein, such that the variant, fragment,derivative, or analog can be activated by cleavage of the proproteinportion to produce an active polypeptide.

Optimizing Codons to Achieve High Levels of Protein Expression in HostCells

The invention provides methods for modifying amylase-encoding nucleicacids to modify codon usage. In one aspect, the invention providesmethods for modifying codons in a nucleic acid encoding an amylase toincrease or decrease its expression in a host cell. The invention alsoprovides nucleic acids encoding an amylase modified to increase itsexpression in a host cell, amylase so modified, and methods of makingthe modified amylases. The method comprises identifying a“non-preferred” or a “less preferred” codon in amylase-encoding nucleicacid and replacing one or more of these non-preferred or less preferredcodons with a “preferred codon” encoding the same amino acid as thereplaced codon and at least one non-preferred or less preferred codon inthe nucleic acid has been replaced by a preferred codon encoding thesame amino acid. A preferred codon is a codon over-represented in codingsequences in genes in the host cell and a non-preferred or lesspreferred codon is a codon under-represented in coding sequences ingenes in the host cell.

Host cells for expressing the nucleic acids, expression cassettes andvectors of the invention include bacteria, yeast, fungi, plant cells,insect cells and mammalian cells. Thus, the invention provides methodsfor optimizing codon usage in all of these cells, codon-altered nucleicacids and polypeptides made by the codon-altered nucleic acids.Exemplary host cells include gram negative bacteria, such as Escherichiacoli and Pseudomonas fluorescens; gram positive bacteria, such asStreptomyces diversa, Lactobacillus gasseri, Lactococcus lactis,Lactococcus cremoris, Bacillus subtilis. Exemplary host cells alsoinclude eukaryotic organisms, e.g., various yeast, such as Saccharomycessp., including Saccharomyces cerevisiae, Schizosaccharomyces pombe,Pichia pastoris, and Kluyveromyces lactis, Hansenula polymorpha,Aspergillus niger, and mammalian cells and cell lines and insect cellsand cell lines. Thus, the invention also includes nucleic acids andpolypeptides optimized for expression in these organisms and species.

For example, the codons of a nucleic acid encoding an amylase isolatedfrom a bacterial cell are modified such that the nucleic acid isoptimally expressed in a bacterial cell different from the bacteria fromwhich the amylase was derived, a yeast, a fungi, a plant cell, an insectcell or a mammalian cell. Methods for optimizing codons are well knownin the art, see, e.g., U.S. Pat. No. 5,795,737; Baca (2000) Int. J.Parasitol. 30:113-118; Hale (1998) Protein Expr. Purif. 12:185-188;Narum (2001) Infect. Immun 69:7250-7253. See also Narum (2001) Infect.Immun 69:7250-7253, describing optimizing codons in mouse systems;Outchkourov (2002) Protein Expr. Purif. 24:18-24, describing optimizingcodons in yeast; Feng (2000) Biochemistry 39:15399-15409, describingoptimizing codons in E. coli; Humphreys (2000) Protein Expr. Purif.20:252-264, describing optimizing codon usage that affects secretion inE. coli.

Transgenic Non-Human Animals

The invention provides transgenic non-human animals comprising a nucleicacid, a polypeptide (e.g., an amylase), an expression cassette or vectoror a transfected or transformed cell of the invention. The inventionalso provides methods of making and using these transgenic non-humananimals.

The transgenic non-human animals can be, e.g., goats, rabbits, sheep,pigs, cows, rats and mice, comprising the nucleic acids of theinvention. These animals can be used, e.g., as in vivo models to studyamylase activity, or, as models to screen for agents that change theamylase activity in vivo. The coding sequences for the polypeptides tobe expressed in the transgenic non-human animals can be designed to beconstitutive, or, under the control of tissue-specific,developmental-specific or inducible transcriptional regulatory factors.Transgenic non-human animals can be designed and generated using anymethod known in the art; see, e.g., U.S. Pat. Nos. 6,211,428; 6,187,992;6,156,952; 6,118,044; 6,111,166; 6,107,541; 5,959,171; 5,922,854;5,892,070; 5,880,327; 5,891,698; 5,639,940; 5,573,933; 5,387,742;5,087,571, describing making and using transformed cells and eggs andtransgenic mice, rats, rabbits, sheep, pigs and cows. See also, e.g.,Pollock (1999) J. Immunol. Methods 231:147-157, describing theproduction of recombinant proteins in the milk of transgenic dairyanimals; Baguisi (1999) Nat. Biotechnol. 17:456-461, demonstrating theproduction of transgenic goats. U.S. Pat. No. 6,211,428, describesmaking and using transgenic non-human mammals which express in theirbrains a nucleic acid construct comprising a DNA sequence. U.S. Pat. No.5,387,742, describes injecting cloned recombinant or synthetic DNAsequences into fertilized mouse eggs, implanting the injected eggs inpseudo-pregnant females, and growing to term transgenic mice whose cellsexpress proteins related to the pathology of Alzheimer's disease. U.S.Pat. No. 6,187,992, describes making and using a transgenic mouse whosegenome comprises a disruption of the gene encoding amyloid precursorprotein (APP).

“Knockout animals” can also be used to practice the methods of theinvention. For example, in one aspect, the transgenic or modifiedanimals of the invention comprise a “knockout animal,” e.g., a “knockoutmouse,” engineered not to express an endogenous gene, which is replacedwith a gene expressing an amylase of the invention, or, a fusion proteincomprising an amylase of the invention.

Transgenic Plants and Seeds

The invention provides transgenic plants and seeds comprising a nucleicacid, a polypeptide (e.g., an amylase, such as an alpha amylase), anexpression cassette or vector or a transfected or transformed cell ofthe invention. The invention also provides plant products, e.g., oils,seeds, leaves, extracts and the like, comprising a nucleic acid and/or apolypeptide (e.g., an amylase, such as an alpha amylase) of theinvention. The transgenic plant can be dicotyledonous (a dicot) ormonocotyledonous (a monocot). The invention also provides methods ofmaking and using these transgenic plants and seeds. The transgenic plantor plant cell expressing a polypeptide of the present invention may beconstructed in accordance with any method known in the art. See, forexample, U.S. Pat. No. 6,309,872.

Nucleic acids and expression constructs of the invention can beintroduced into a plant cell by any means. For example, nucleic acids orexpression constructs can be introduced into the genome of a desiredplant host, or, the nucleic acids or expression constructs can beepisomes. Introduction into the genome of a desired plant can be suchthat the host's α-amylase production is regulated by endogenoustranscriptional or translational control elements. The invention alsoprovides “knockout plants” where insertion of gene sequence by, e.g.,homologous recombination, has disrupted the expression of the endogenousgene. Means to generate “knockout” plants are well-known in the art,see, e.g., Strepp (1998) Proc Natl. Acad. Sci. USA 95:4368-4373; Miao(1995) Plant J 7:359-365. See discussion on transgenic plants, below.

The nucleic acids of the invention can be used to confer desired traitson essentially any plant, e.g., on starch-producing plants, such aspotato, wheat, rice, barley, and the like. Nucleic acids of theinvention can be used to manipulate metabolic pathways of a plant inorder to optimize or alter host's expression of α-amylase. The canchange the ratio of starch/sugar conversion in a plant. This canfacilitate industrial processing of a plant. Alternatively,alpha-amylases of the invention can be used in production of atransgenic plant to produce a compound not naturally produced by thatplant. This can lower production costs or create a novel product.

In one aspect, the first step in production of a transgenic plantinvolves making an expression construct for expression in a plant cell.These techniques are well known in the art. They can include selectingand cloning a promoter, a coding sequence for facilitating efficientbinding of ribosomes to mRNA and selecting the appropriate geneterminator sequences. One exemplary constitutive promoter is CaMV35S,from the cauliflower mosaic virus, which generally results in a highdegree of expression in plants. Other promoters are more specific andrespond to cues in the plant's internal or external environment. Anexemplary light-inducible promoter is the promoter from the cab gene,encoding the major chlorophyll a/b binding protein.

In one aspect, the nucleic acid is modified to achieve greaterexpression in a plant cell. For example, a sequence of the invention islikely to have a higher percentage of A-T nucleotide pairs compared tothat seen in a plant, some of which prefer G-C nucleotide pairs.Therefore, A-T nucleotides in the coding sequence can be substitutedwith G-C nucleotides without significantly changing the amino acidsequence to enhance production of the gene product in plant cells.

Selectable marker gene can be added to the gene construct in order toidentify plant cells or tissues that have successfully integrated thetransgene. This may be necessary because achieving incorporation andexpression of genes in plant cells is a rare event, occurring in just afew percent of the targeted tissues or cells. Selectable marker genesencode proteins that provide resistance to agents that are normallytoxic to plants, such as antibiotics or herbicides. Only plant cellsthat have integrated the selectable marker gene will survive when grownon a medium containing the appropriate antibiotic or herbicide. As forother inserted genes, marker genes also require promoter and terminationsequences for proper function.

In one aspect, making transgenic plants or seeds comprises incorporatingsequences of the invention and, optionally, marker genes into a targetexpression construct (e.g., a plasmid), along with positioning of thepromoter and the terminator sequences. This can involve transferring themodified gene into the plant through a suitable method. For example, aconstruct may be introduced directly into the genomic DNA of the plantcell using techniques such as electroporation and microinjection ofplant cell protoplasts, or the constructs can be introduced directly toplant tissue using ballistic methods, such as DNA particle bombardment.For example, see, e.g., Christou (1997) Plant Mol. Biol. 35:197-203;Pawlowski (1996) Mol. Biotechnol. 6:17-30; Klein (1987) Nature327:70-73; Takumi (1997) Genes Genet. Syst. 72:63-69, discussing use ofparticle bombardment to introduce transgenes into wheat; and Adam (1997)supra, for use of particle bombardment to introduce YACs into plantcells. For example, Rinehart (1997) supra, used particle bombardment togenerate transgenic cotton plants. Apparatus for accelerating particlesis described U.S. Pat. No. 5,015,580; and, the commercially availableBioRad (Biolistics) PDS-2000 particle acceleration instrument; see also,John, U.S. Pat. No. 5,608,148; and Ellis, U.S. Pat. No. 5,681,730,describing particle-mediated transformation of gymnosperms.

In one aspect, protoplasts can be immobilized and injected with anucleic acids, e.g., an expression construct. Although plantregeneration from protoplasts is not easy with cereals, plantregeneration is possible in legumes using somatic embryogenesis fromprotoplast derived callus. Organized tissues can be transformed withnaked DNA using gene gun technique, where DNA is coated on tungstenmicroprojectiles, shot 1/100th the size of cells, which carry the DNAdeep into cells and organelles. Transformed tissue is then induced toregenerate, usually by somatic embryogenesis. This technique has beensuccessful in several cereal species including maize and rice.

Nucleic acids, e.g., expression constructs, can also be introduced in toplant cells using recombinant viruses. Plant cells can be transformedusing viral vectors, such as, e.g., tobacco mosaic virus derived vectors(Rouwendal (1997) Plant Mol. Biol. 33:989-999), see Porta (1996) “Use ofviral replicons for the expression of genes in plants,” Mol. Biotechnol.5:209-221.

Alternatively, nucleic acids, e.g., an expression construct, can becombined with suitable T-DNA flanking regions and introduced into aconventional Agrobacterium tumefaciens host vector. The virulencefunctions of the Agrobacterium tumefaciens host will direct theinsertion of the construct and adjacent marker into the plant cell DNAwhen the cell is infected by the bacteria. Agrobacteriumtumefaciens-mediated transformation techniques, including disarming anduse of binary vectors, are well described in the scientific literature.See, e.g., Horsch (1984) Science 233:496-498; Fraley (1983) Proc. Natl.Acad. Sci. USA 80:4803 (1983); Gene Transfer to Plants, Potrykus, ed.(Springer-Verlag, Berlin 1995). The DNA in an A. tumefaciens cell iscontained in the bacterial chromosome as well as in another structureknown as a Ti (tumor-inducing) plasmid. The Ti plasmid contains astretch of DNA termed T-DNA (˜20 kb long) that is transferred to theplant cell in the infection process and a series of vir (virulence)genes that direct the infection process. A. tumefaciens can only infecta plant through wounds: when a plant root or stem is wounded it givesoff certain chemical signals, in response to which, the vir genes of A.tumefaciens become activated and direct a series of events necessary forthe transfer of the T-DNA from the Ti plasmid to the plant's chromosome.The T-DNA then enters the plant cell through the wound. One speculationis that the T-DNA waits until the plant DNA is being replicated ortranscribed, then inserts itself into the exposed plant DNA. In order touse A. tumefaciens as a transgene vector, the tumor-inducing section ofT-DNA have to be removed, while retaining the T-DNA border regions andthe vir genes. The transgene is then inserted between the T-DNA borderregions, where it is transferred to the plant cell and becomesintegrated into the plant's chromosomes.

The invention provides for the transformation of monocotyledonous plantsusing the nucleic acids of the invention, including important cereals,see Hiei (1997) Plant Mol. Biol. 35:205-218. See also, e.g., Horsch,Science (1984) 233:496; Fraley (1983) Proc. Natl Acad. Sci USA 80:4803;Thykjaer (1997) supra; Park (1996) Plant Mol. Biol. 32:1135-1148,discussing T-DNA integration into genomic DNA. See also D'Halluin, U.S.Pat. No. 5,712,135, describing a process for the stable integration of aDNA comprising a gene that is functional in a cell of a cereal, or othermonocotyledonous plant.

In one aspect, the third step can involve selection and regeneration ofwhole plants capable of transmitting the incorporated target gene to thenext generation. Such regeneration techniques rely on manipulation ofcertain phytohormones in a tissue culture growth medium, typicallyrelying on a biocide and/or herbicide marker that has been introducedtogether with the desired nucleotide sequences. Plant regeneration fromcultured protoplasts is described in Evans et al., Protoplasts Isolationand Culture, Handbook of Plant Cell Culture, pp. 124-176, MacMillilanPublishing Company, New York, 1983; and Binding, Regeneration of Plants,Plant Protoplasts, pp. 21-73, CRC Press, Boca Raton, 1985. Regenerationcan also be obtained from plant callus, explants, organs, or partsthereof. Such regeneration techniques are described generally in Klee(1987) Ann. Rev. of Plant Phys. 38:467-486. To obtain whole plants fromtransgenic tissues such as immature embryos, they can be grown undercontrolled environmental conditions in a series of media containingnutrients and hormones, a process known as tissue culture. Once wholeplants are generated and produce seed, evaluation of the progeny begins.

After the expression cassette is stably incorporated in transgenicplants, it can be introduced into other plants by sexual crossing. Anyof a number of standard breeding techniques can be used, depending uponthe species to be crossed. Since transgenic expression of the nucleicacids of the invention leads to phenotypic changes, plants comprisingthe recombinant nucleic acids of the invention can be sexually crossedwith a second plant to obtain a final product. Thus, the seed of theinvention can be derived from a cross between two transgenic plants ofthe invention, or a cross between a plant of the invention and anotherplant. The desired effects (e.g., expression of the polypeptides of theinvention to produce a plant in which flowering behavior is altered) canbe enhanced when both parental plants express the polypeptides (e.g., anamylase, such as an alpha amylase) of the invention. The desired effectscan be passed to future plant generations by standard propagation means.

The nucleic acids and polypeptides of the invention are expressed in orinserted in any plant or seed. Transgenic plants of the invention can bedicotyledonous or monocotyledonous. Examples of monocot transgenicplants of the invention are grasses, such as meadow grass (blue grass,Poa), forage grass such as festuca, lolium, temperate grass, such asAgrostis, and cereals, e.g., wheat, oats, rye, barley, rice, sorghum,and maize (corn). Examples of dicot transgenic plants of the inventionare tobacco, legumes, such as lupins, potato, sugar beet, pea, bean andsoybean, and cruciferous plants (family Brassicaceae), such ascauliflower, rape seed, and the closely related model organismArabidopsis thaliana. Thus, the transgenic plants and seeds of theinvention include a broad range of plants, including, but not limitedto, species from the genera Anacardium, Arachis, Asparagus, Atropa,Avena, Brassica, Citrus, Citrullus, Capsicum, Carthamus, Cocos, Coffea,Cucumis, Cucurbita, Daucus, Elaeis, Fragaria, Glycine, Gossypium,Helianthus, Heterocallis, Hordeum, Hyoscyamus, Lactuca, Linum, Lolium,Lupinus, Lycopersicon, Malus, Manihot, Majorana, Medicago, Nicotiana,Olea, Oryza, Panieum, Pannisetum, Persea, Phaseolus, Pistachia, Pisum,Pyrus, Prunus, Raphanus, Ricinus, Secale, Senecio, Sinapis, Solanum,Sorghum, Theobromus, Trigonella, Triticum, Vicia, Vitis, Vigna, and Zea.

In alternative embodiments, the nucleic acids of the invention areexpressed in plants which contain fiber cells, including, e.g., cotton,silk cotton tree (Kapok, Ceiba pentandra), desert willow, creosote bush,winterfat, balsa, ramie, kenaf, hemp, roselle, jute, sisal abaca andflax. In alternative embodiments, the transgenic plants of the inventioncan be members of the genus Gossypium, including members of anyGossypium species, such as G. arboreum; G. herbaceum, G. barbadense, andG. hirsutum.

The invention also provides for transgenic plants to be used forproducing large amounts of the polypeptides (e.g., an amylase, such asan alpha amylase) of the invention. For example, see Palmgren (1997)Trends Genet. 13:348; Chong (1997) Transgenic Res. 6:289-296 (producinghuman milk protein beta-casein in transgenic potato plants using anauxin-inducible, bidirectional mannopine synthase (mas 1′,2′) promoterwith Agrobacterium tumefaciens-mediated leaf disc transformationmethods).

Using known procedures, one of skill can screen for plants of theinvention by detecting the increase or decrease of transgene mRNA orprotein in transgenic plants. Means for detecting and quantitation ofmRNAs or proteins are well known in the art.

Polypeptides and Peptides

In one aspect, the invention provides isolated or recombinantpolypeptides having a sequence identity (e.g., at least about 50%, 51%,52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%) sequenceidentity) to an exemplary sequence of the invention, e.g., SEQ ID NO:2and subsequences thereof and variants thereof. In one aspect, thepolypeptide has an amylase activity, e.g., an alpha amylase activity.

The identity can be over the full length of the polypeptide, or, theidentity can be over a region of at least about 50, 60, 70, 80, 90, 100,150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700 or moreresidues. Polypeptides of the invention can also be shorter than thefull length of exemplary polypeptides. In alternative aspects, theinvention provides polypeptides (peptides, fragments) ranging in sizebetween about 5 and the full length of a polypeptide, e.g., an enzyme,such as an amylase; exemplary sizes being of about 5, 10, 15, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 125, 150, 175,200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, or more residues,e.g., contiguous residues of an exemplary amylase of the invention.Peptides of the invention can be useful as, e.g., labeling probes,antigens, toleragens, motifs, amylase active sites.

Polypeptides and peptides of the invention can be isolated from naturalsources, be synthetic, or be recombinantly generated polypeptides.Peptides and proteins can be recombinantly expressed in vitro or invivo. The peptides and polypeptides of the invention can be made andisolated using any method known in the art. Polypeptide and peptides ofthe invention can also be synthesized, whole or in part, using chemicalmethods well known in the art. See e.g., Caruthers (1980) Nucleic AcidsRes. Symp. Ser. 215-223; Horn (1980) Nucleic Acids Res. Symp. Ser.225-232; Banga, A. K., Therapeutic Peptides and Proteins, Formulation,Processing and Delivery Systems (1995) Technomic Publishing Co.,Lancaster, Pa. For example, peptide synthesis can be performed usingvarious solid-phase techniques (see e.g., Roberge (1995) Science269:202; Merrifield (1997) Methods Enzymol. 289:3-13) and automatedsynthesis may be achieved, e.g., using the ABI 431A Peptide Synthesizer(Perkin Elmer) in accordance with the instructions provided by themanufacturer.

The peptides and polypeptides of the invention can also be glycosylated.The glycosylation can be added post-translationally either chemically orby cellular biosynthetic mechanisms, wherein the later incorporates theuse of known glycosylation motifs, which can be native to the sequenceor can be added as a peptide or added in the nucleic acid codingsequence. The glycosylation can be O-linked or N-linked.

The peptides and polypeptides of the invention, as defined above,include all “mimetic” and “peptidomimetic” forms. The terms “mimetic”and “peptidomimetic” refer to a synthetic chemical compound which hassubstantially the same structural and/or functional characteristics ofthe polypeptides of the invention. The mimetic can be either entirelycomposed of synthetic, non-natural analogues of amino acids, or, is achimeric molecule of partly natural peptide amino acids and partlynon-natural analogs of amino acids. The mimetic can also incorporate anyamount of natural amino acid conservative substitutions as long as suchsubstitutions also do not substantially alter the mimetic's structureand/or activity. As with polypeptides of the invention which areconservative variants, routine experimentation will determine whether amimetic is within the scope of the invention, i.e., that its structureand/or function is not substantially altered. Thus, in one aspect, amimetic composition is within the scope of the invention if it has anamylase activity.

Polypeptide mimetic compositions of the invention can contain anycombination of non-natural structural components. In alternative aspect,mimetic compositions of the invention include one or all of thefollowing three structural groups: a) residue linkage groups other thanthe natural amide bond (“peptide bond”) linkages; b) non-naturalresidues in place of naturally occurring amino acid residues; or c)residues which induce secondary structural mimicry, i.e., to induce orstabilize a secondary structure, e.g., a beta turn, gamma turn, betasheet, alpha helix conformation, and the like. For example, apolypeptide of the invention can be characterized as a mimetic when allor some of its residues are joined by chemical means other than naturalpeptide bonds. Individual peptidomimetic residues can be joined bypeptide bonds, other chemical bonds or coupling means, such as, e.g.,glutaraldehyde, N-hydroxysuccinimide esters, bifunctional maleimides,N,N′-dicyclohexylcarbodiimide (DCC) or N,N′-diisopropylcarbodiimide(DIC). Linking groups that can be an alternative to the traditionalamide bond (“peptide bond”) linkages include, e.g., ketomethylene (e.g.,—C(═O)—CH₂— for —C(═O)—NH—), aminomethylene (CH₂—NH), ethylene, olefin(CH═CH), ether (CH₂—O), thioether (CH₂—S), tetrazole (CN₄—), thiazole,retroamide, thioamide, or ester (see, e.g., Spatola (1983) in Chemistryand Biochemistry of Amino Acids, Peptides and Proteins, Vol. 7, pp267-357, “Peptide Backbone Modifications,” Marcell Dekker, N.Y.).

A polypeptide of the invention can also be characterized as a mimetic bycontaining all or some non-natural residues in place of naturallyoccurring amino acid residues. Non-natural residues are well describedin the scientific and patent literature; a few exemplary non-naturalcompositions useful as mimetics of natural amino acid residues andguidelines are described below. Mimetics of aromatic amino acids can begenerated by replacing by, e.g., D- or L-naphylalanine; D- orL-phenylglycine; D- or L-2 thieneylalanine; D- or L-1, -2,3-, or4-pyreneylalanine; D- or L-3 thieneylalanine; D- orL-(2-pyridinyl)-alanine; D- or L-(3-pyridinyl)-alanine; D- orL-(2-pyrazinyl)-alanine; D- or L-(4-isopropyl)-phenylglycine;D-(trifluoromethyl)-phenylglycine; D-(trifluoromethyl)-phenylalanine;D-p-fluoro-phenylalanine; D- or L-p-biphenylphenylalanine; D- orL-p-methoxy-biphenylphenylalanine; D- or L-2-indole(alkyl)alanines; and,D- or L-alkylainines, where alkyl can be substituted or unsubstitutedmethyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, iso-butyl,sec-isotyl, iso-pentyl, or a non-acidic amino acids. Aromatic rings of anon-natural amino acid include, e.g., thiazolyl, thiophenyl, pyrazolyl,benzimidazolyl, naphthyl, furanyl, pyrrolyl, and pyridyl aromatic rings.

Mimetics of acidic amino acids can be generated by substitution by,e.g., non-carboxylate amino acids while maintaining a negative charge;(phosphono)alanine; sulfated threonine. Carboxyl side groups (e.g.,aspartyl or glutamyl) can also be selectively modified by reaction withcarbodiimides (R′—N—C—N—R′) such as, e.g.,1-cyclohexyl-3(2-morpholinyl-(4-ethyl) carbodiimide or1-ethyl-3(4-azonia-4,4-dimetholpentyl) carbodiimide. Aspartyl orglutamyl can also be converted to asparaginyl and glutaminyl residues byreaction with ammonium ions. Mimetics of basic amino acids can begenerated by substitution with, e.g., (in addition to lysine andarginine) the amino acids ornithine, citrulline, or (guanidino)-aceticacid, or (guanidino)alkyl-acetic acid, where alkyl is defined above.Nitrile derivative (e.g., containing the CN-moiety in place of COOH) canbe substituted for asparagine or glutamine. Asparaginyl and glutaminylresidues can be deaminated to the corresponding aspartyl or glutamylresidues. Arginine residue mimetics can be generated by reacting arginylwith, e.g., one or more conventional reagents, including, e.g.,phenylglyoxal, 2,3-butanedione, 1,2-cyclo-hexanedione, or ninhydrin,preferably under alkaline conditions. Tyrosine residue mimetics can begenerated by reacting tyrosyl with, e.g., aromatic diazonium compoundsor tetranitromethane. N-acetylimidizol and tetranitromethane can be usedto form O-acetyl tyrosyl species and 3-nitro derivatives, respectively.Cysteine residue mimetics can be generated by reacting cysteinylresidues with, e.g., alpha-haloacetates such as 2-chloroacetic acid orchloroacetamide and corresponding amines; to give carboxymethyl orcarboxyamidomethyl derivatives. Cysteine residue mimetics can also begenerated by reacting cysteinyl residues with, e.g.,bromo-trifluoroacetone, alpha-bromo-beta-(5-imidozoyl) propionic acid;chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide;methyl 2-pyridyl disulfide; p-chloromercuribenzoate; 2-chloromercuri-4nitrophenol; or, chloro-7-nitrobenzo-oxa-1,3-diazole. Lysine mimeticscan be generated (and amino terminal residues can be altered) byreacting lysinyl with, e.g., succinic or other carboxylic acidanhydrides. Lysine and other alpha-amino-containing residue mimetics canalso be generated by reaction with imidoesters, such as methylpicolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride,trinitro-benzenesulfonic acid, O-methylisourea, 2,4, pentanedione, andtransamidase-catalyzed reactions with glyoxylate. Mimetics of methioninecan be generated by reaction with, e.g., methionine sulfoxide. Mimeticsof proline include, e.g., pipecolic acid, thiazolidine carboxylic acid,3- or 4-hydroxy proline, dehydroproline, 3- or 4-methylproline, or3,3,-dimethylproline. Histidine residue mimetics can be generated byreacting histidyl with, e.g., diethylprocarbonate or para-bromophenacylbromide. Other mimetics include, e.g., those generated by hydroxylationof proline and lysine; phosphorylation of the hydroxyl groups of serylor threonyl residues; methylation of the alpha-amino groups of lysine,arginine and histidine; acetylation of the N-terminal amine; methylationof main chain amide residues or substitution with N-methyl amino acids;or amidation of C-terminal carboxyl groups.

A residue, e.g., an amino acid, of a polypeptide of the invention canalso be replaced by an amino acid (or peptidomimetic residue) of theopposite chirality. Thus, any amino acid naturally occurring in theL-configuration (which can also be referred to as the R or S, dependingupon the structure of the chemical entity) can be replaced with theamino acid of the same chemical structural type or a peptidomimetic, butof the opposite chirality, referred to as the D-amino acid, but also canbe referred to as the R- or S-form.

The invention also provides methods for modifying the polypeptides ofthe invention by either natural processes, such as post-translationalprocessing (e.g., phosphorylation, acylation, etc), or by chemicalmodification techniques, and the resulting modified polypeptides.Modifications can occur anywhere in the polypeptide, including thepeptide backbone, the amino acid side-chains and the amino or carboxyltermini. It will be appreciated that the same type of modification maybe present in the same or varying degrees at several sites in a givenpolypeptide. Also a given polypeptide may have many types ofmodifications. Modifications include acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of a phosphatidylinositol, cross-linkingcyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristolyation, oxidation,pegylation, proteolytic processing, phosphorylation, prenylation,racemization, selenoylation, sulfation, and transfer-RNA mediatedaddition of amino acids to protein such as arginylation. See, e.g.,Creighton, T. E., Proteins—Structure and Molecular Properties 2nd Ed.,W.H. Freeman and Company, New York (1993); Posttranslational CovalentModification of Proteins, B. C. Johnson, Ed., Academic Press, New York,pp. 1-12 (1983).

Solid-phase chemical peptide synthesis methods can also be used tosynthesize the polypeptide or fragments of the invention. Such methodhave been known in the art since the early 1960's (Merrifield, R. B., J.Am. Chem. Soc., 85:2149-2154, 1963) (See also Stewart, J. M. and Young,J. D., Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Co.,Rockford, Ill., pp. 11-12)) and have recently been employed incommercially available laboratory peptide design and synthesis kits(Cambridge Research Biochemicals). Such commercially availablelaboratory kits have generally utilized the teachings of H. M. Geysen etal, Proc. Natl. Acad. Sci., USA, 81:3998 (1984) and provide forsynthesizing peptides upon the tips of a multitude of “rods” or “pins”all of which are connected to a single plate. When such a system isutilized, a plate of rods or pins is inverted and inserted into a secondplate of corresponding wells or reservoirs, which contain solutions forattaching or anchoring an appropriate amino acid to the pin's or rod'stips. By repeating such a process step, i.e., inverting and insertingthe rod's and pin's tips into appropriate solutions, amino acids arebuilt into desired peptides. In addition, a number of available FMOCpeptide synthesis systems are available. For example, assembly of apolypeptide or fragment can be carried out on a solid support using anApplied Biosystems, Inc. Model 431A™ automated peptide synthesizer. Suchequipment provides ready access to the peptides of the invention, eitherby direct synthesis or by synthesis of a series of fragments that can becoupled using other known techniques.

The invention provides novel amylases (e.g., alpha amylases), includingthe exemplary enzymes of the invention, nucleic acids encoding them,antibodies that bind them, and methods for making and using them. In oneaspect, the polypeptides of the invention have an amylase activity, asdescribed herein, including, e.g., the ability to hydrolyze starchesinto sugars. In one aspect, the polypeptides of the invention have analpha amylase activity. In alternative aspects, the amylases of theinvention have activities that have been modified from those of theexemplary amylases described herein.

The invention includes amylases of the invention with and without signalsequences (including signal sequence of the invention, or other signalsequences) and the signal sequences themselves. The invention alsoinclude polypeptides (e.g., fusion proteins) comprising a signalsequence of the invention. The polypeptide comprising a signal sequenceof the invention can be an amylase of the invention or another amylaseor another enzyme or other polypeptide.

The invention includes immobilized amylases, anti-amylase antibodies andfragments thereof. The invention provides methods for inhibiting amylaseactivity, e.g., using dominant negative mutants or anti-amylaseantibodies of the invention. The invention includes heterocomplexes,e.g., fusion proteins, heterodimers, etc., comprising the amylases ofthe invention.

In one aspect, amylases (e.g., alpha amylases) of the inventionhydrolyze internal polysaccharide bonds, e.g., α-1,4- and 1,6-glucosidicbonds in starch to produce smaller molecular weight maltodextrines. Inone aspect, this hydrolysis is largely at random. Thus, the inventionprovides methods for producing smaller molecular weight maltodextrines.

Amylases of the invention can be used in laboratory and industrialsettings to hydrolyze starch or any maltodextrine-comprising compoundfor a variety of purposes. These amylases can be used alone to providespecific hydrolysis or can be combined with other amylases to provide a“cocktail” with a broad spectrum of activity. Exemplary uses include theremoval or partial or complete hydrolysis of starch or anymaltodextrine-comprising compound from biological, food, animal feed,pharmaceutical or industrial samples.

For example, the amylases of the present invention can be formulated inlaundry detergents to aid in the removal of starch-containing stains.Amylases of the invention can be used as cleaning agents in detergentmatrices (see industrial applications below). The amylases of thepresent invention can be used in the initial stages (liquefaction) ofstarch processing, in wet corn milling, in alcohol production, in thetextile industry for starch desizing, in baking applications, in thebeverage industry, in oilfields in drilling processes; in inking ofrecycled paper; and in animal feed.

Amylases of the invention can have an amylase activity under variousconditions, e.g., extremes in pH and/or temperature, oxidizing agents,and the like. The invention provides methods leading to alternativeamylase preparations with different catalytic efficiencies andstabilities, e.g., towards temperature, oxidizing agents and changingwash conditions. In one aspect, amylase variants can be produced usingtechniques of site-directed mutagenesis and/or random mutagenesis. Inone aspect, directed evolution can be used to produce a great variety ofamylase variants with alternative specificities and stability.

The proteins of the invention are also useful as research reagents toidentify amylase modulators, e.g., activators or inhibitors of amylaseactivity. Briefly, test samples (compounds, broths, extracts, and thelike) are added to amylase assays to determine their ability to inhibitsubstrate cleavage Inhibitors identified in this way can be used inindustry and research to reduce or prevent undesired proteolysis. Aswith amylases, inhibitors can be combined to increase the spectrum ofactivity.

The invention also provides methods of discovering new amylases usingthe nucleic acids, polypeptides and antibodies of the invention. In oneaspect, lambda phage libraries are screened for expression-baseddiscovery of amylases. In one aspect, the invention uses lambda phagelibraries in screening to allow detection of toxic clones; improvedaccess to substrate; reduced need for engineering a host, by-passing thepotential for any bias resulting from mass excision of the library; and,faster growth at low clone densities. Screening of lambda phagelibraries can be in liquid phase or in solid phase. In one aspect, theinvention provides screening in liquid phase. This gives a greaterflexibility in assay conditions; additional substrate flexibility;higher sensitivity for weak clones; and ease of automation over solidphase screening.

The invention provides screening methods using the proteins and nucleicacids of the invention and robotic automation to enable the execution ofmany thousands of biocatalytic reactions and screening assays in a shortperiod of time, e.g., per day, as well as ensuring a high level ofaccuracy and reproducibility (see discussion of arrays, below). As aresult, a library of derivative compounds can be produced in a matter ofweeks. For further teachings on modification of molecules, includingsmall molecules, see PCT/US94/09174.

The present invention includes amylase enzymes which are non-naturallyoccurring carbonyl hydrolase variants (e.g., amylase variants) having adifferent proteolytic activity, stability, substrate specificity, pHprofile and/or performance characteristic as compared to the precursorcarbonyl hydrolase from which the amino acid sequence of the variant isderived. Specifically, such amylase variants have an amino acid sequencenot found in nature, which is derived by substitution of a plurality ofamino acid residues of a precursor amylase with different amino acids.The precursor amylase may be a naturally-occurring amylase or arecombinant amylase. The useful amylase variants encompass thesubstitution of any of the naturally occurring L-amino acids at thedesignated amino acid residue positions.

Amylase Signal Sequences

The invention provides amylase signal sequences and nucleic acidsencoding these signal sequences, e.g., the amino terminal 13 to 36residues of SEQ ID NO:2. The amylase signal sequences of the inventioncan be isolated peptides, or, sequences joined to another amylase or anon-amylase polypeptide, e.g., as a fusion protein. In one aspect, theinvention provides polypeptides comprising amylase signal sequences ofthe invention. In one aspect, polypeptides comprising amylase signalsequences of the invention comprise sequences heterologous to an amylaseof the invention (e.g., a fusion protein comprising an amylase signalsequence of the invention and sequences from another amylase or anon-amylase protein). In one aspect, the invention provides amylases ofthe invention with heterologous signal sequences, e.g., sequences with ayeast signal sequence. For example, an amylase of the inventioncomprising a heterologous signal sequence in a vectors, e.g., a pPICseries vector (Invitrogen, Carlsbad, Calif.).

In one aspect, the signal sequences of the invention are identifiedfollowing identification of novel amylase polypeptides. The pathways bywhich proteins are sorted and transported to their proper cellularlocation are often referred to as protein targeting pathways. One of themost important elements in all of these targeting systems is a shortamino acid sequence at the amino terminus of a newly synthesizedpolypeptide called the signal sequence. This signal sequence directs aprotein to its appropriate location in the cell and is removed duringtransport or when the protein reaches its final destination. Mostlysosomal, membrane, or secreted proteins have an amino-terminal signalsequence that marks them for translocation into the lumen of theendoplasmic reticulum. More than 100 signal sequences for proteins inthis group have been determined. The signal sequences can vary in lengthfrom 13 to 36 amino acid residues. Various methods of recognition ofsignal sequences are known to those of skill in the art. For example, inone aspect, novel amylase signal peptides are identified by a methodreferred to as SignalP. SignalP uses a combined neural network whichrecognizes both signal peptides and their cleavage sites. (Nielsen, etal., “Identification of prokaryotic and eukaryotic signal peptides andprediction of their cleavage sites.” Protein Engineering, vol. 10, no.1, p. 1-6 (1997).

It should be understood that in some aspects amylases of the inventionmay not have signal sequences. In one aspect, the invention provides theamylases of the invention lacking all or part of a signal sequence, e.g.the signal sequences of the invention. In one aspect, the inventionprovides a nucleic acid sequence encoding a signal sequence from oneamylase operably linked to a nucleic acid sequence of a differentamylase or, optionally, a signal sequence from a non-amylase protein maybe desired.

Hybrid Amylases and Peptide Libraries

In one aspect, the invention provides hybrid amylases and fusionproteins, including peptide libraries, comprising sequences of theinvention. The peptide libraries of the invention can be used to isolatepeptide modulators (e.g., activators or inhibitors) of targets, such asamylase substrates, receptors, enzymes. The peptide libraries of theinvention can be used to identify formal binding partners of targets,such as ligands, e.g., cytokines, hormones and the like.

In one aspect, the fusion proteins of the invention (e.g., the peptidemoiety) are conformationally stabilized (relative to linear peptides) toallow a higher binding affinity for targets. The invention providesfusions of amylases of the invention and other peptides, including knownand random peptides. They can be fused in such a manner that thestructure of the amylases is not significantly perturbed and the peptideis metabolically or structurally conformationally stabilized. Thisallows the creation of a peptide library that is easily monitored bothfor its presence within cells and its quantity.

Amino acid sequence variants of the invention can be characterized by apredetermined nature of the variation, a feature that sets them apartfrom a naturally occurring form, e.g., an allelic or interspeciesvariation of an amylase sequence. In one aspect, the variants of theinvention exhibit the same qualitative biological activity as thenaturally occurring analogue. Alternatively, the variants can beselected for having modified characteristics. In one aspect, while thesite or region for introducing an amino acid sequence variation ispredetermined, the mutation per se need not be predetermined. Forexample, in order to optimize the performance of a mutation at a givensite, random mutagenesis may be conducted at the target codon or regionand the expressed amylase variants screened for the optimal combinationof desired activity. Techniques for making substitution mutations atpredetermined sites in DNA having a known sequence are well known, asdiscussed herein for example, M13 primer mutagenesis and PCRmutagenesis. Screening of the mutants can be done using assays ofproteolytic activities. In alternative aspects, amino acid substitutionscan be single residues; insertions can be on the order of from about 1to 20 amino acids, although considerably larger insertions can be done.Deletions can range from about 1 to about 20, 30, 40, 50, 60, 70residues or more. To obtain a final derivative with the optimalproperties, substitutions, deletions, insertions or any combinationthereof may be used. Generally, these changes are done on a few aminoacids to minimize the alteration of the molecule. However, largerchanges may be tolerated in certain circumstances.

The invention provides amylases where the structure of the polypeptidebackbone, the secondary or the tertiary structure, e.g., analpha-helical or beta-sheet structure, has been modified. In one aspect,the charge or hydrophobicity has been modified. In one aspect, the bulkof a side chain has been modified. Substantial changes in function orimmunological identity are made by selecting substitutions that are lessconservative. For example, substitutions can be made which moresignificantly affect: the structure of the polypeptide backbone in thearea of the alteration, for example a alpha-helical or a beta-sheetstructure; a charge or a hydrophobic site of the molecule, which can beat an active site; or a side chain. The invention provides substitutionsin polypeptide of the invention where (a) a hydrophilic residues, e.g.seryl or threonyl, is substituted for (or by) a hydrophobic residue,e.g. leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine orproline is substituted for (or by) any other residue; (c) a residuehaving an electropositive side chain, e.g. lysyl, arginyl, or histidyl,is substituted for (or by) an electronegative residue, e.g. glutamyl oraspartyl; or (d) a residue having a bulky side chain, e.g.phenylalanine, is substituted for (or by) one not having a side chain,e.g. glycine. The variants can exhibit the same qualitative biologicalactivity (i.e. amylase activity) although variants can be selected tomodify the characteristics of the amylases as needed.

In one aspect, amylases of the invention comprise epitopes orpurification tags, signal sequences or other fusion sequences, etc. Inone aspect, the amylases of the invention can be fused to a randompeptide to form a fusion polypeptide. By “fused” or “operably linked”herein is meant that the random peptide and the amylase are linkedtogether, in such a manner as to minimize the disruption to thestability of the amylase structure, e.g., it retains amylase activity.The fusion polypeptide (or fusion polynucleotide encoding the fusionpolypeptide) can comprise further components as well, including multiplepeptides at multiple loops.

In one aspect, the peptides and nucleic acids encoding them arerandomized, either fully randomized or they are biased in theirrandomization, e.g. in nucleotide/residue frequency generally or perposition. “Randomized” means that each nucleic acid and peptide consistsof essentially random nucleotides and amino acids, respectively. In oneaspect, the nucleic acids which give rise to the peptides can bechemically synthesized, and thus may incorporate any nucleotide at anyposition. Thus, when the nucleic acids are expressed to form peptides,any amino acid residue may be incorporated at any position. Thesynthetic process can be designed to generate randomized nucleic acids,to allow the formation of all or most of the possible combinations overthe length of the nucleic acid, thus forming a library of randomizednucleic acids. The library can provide a sufficiently structurallydiverse population of randomized expression products to affect aprobabilistically sufficient range of cellular responses to provide oneor more cells exhibiting a desired response. Thus, the inventionprovides an interaction library large enough so that at least one of itsmembers will have a structure that gives it affinity for some molecule,protein, or other factor.

Screening Methodologies and “on-Line” Monitoring Devices

In practicing the methods of the invention, a variety of apparatus andmethodologies can be used to in conjunction with the polypeptides andnucleic acids of the invention, e.g., to screen polypeptides for amylaseactivity, to screen compounds as potential modulators, e.g., activatorsor inhibitors, of an amylase activity, for antibodies that bind to apolypeptide of the invention, for nucleic acids that hybridize to anucleic acid of the invention, to screen for cells expressing apolypeptide of the invention and the like.

Capillary Arrays

Capillary arrays, such as the GIGAMATRIX™, Diversa Corporation, SanDiego, Calif., can be used to in the methods of the invention. Nucleicacids or polypeptides of the invention can be immobilized to or appliedto an array, including capillary arrays. Arrays can be used to screenfor or monitor libraries of compositions (e.g., small molecules,antibodies, nucleic acids, etc.) for their ability to bind to ormodulate the activity of a nucleic acid or a polypeptide of theinvention. Capillary arrays provide another system for holding andscreening samples. For example, a sample screening apparatus can includea plurality of capillaries formed into an array of adjacent capillaries,wherein each capillary comprises at least one wall defining a lumen forretaining a sample. The apparatus can further include interstitialmaterial disposed between adjacent capillaries in the array, and one ormore reference indicia formed within of the interstitial material. Acapillary for screening a sample, wherein the capillary is adapted forbeing bound in an array of capillaries, can include a first walldefining a lumen for retaining the sample, and a second wall formed of afiltering material, for filtering excitation energy provided to thelumen to excite the sample. A polypeptide or nucleic acid, e.g., aligand, can be introduced into a first component into at least a portionof a capillary of a capillary array. Each capillary of the capillaryarray can comprise at least one wall defining a lumen for retaining thefirst component. An air bubble can be introduced into the capillarybehind the first component. A second component can be introduced intothe capillary, wherein the second component is separated from the firstcomponent by the air bubble. A sample of interest can be introduced as afirst liquid labeled with a detectable particle into a capillary of acapillary array, wherein each capillary of the capillary array comprisesat least one wall defining a lumen for retaining the first liquid andthe detectable particle, and wherein the at least one wall is coatedwith a binding material for binding the detectable particle to the atleast one wall. The method can further include removing the first liquidfrom the capillary tube, wherein the bound detectable particle ismaintained within the capillary, and introducing a second liquid intothe capillary tube. The capillary array can include a plurality ofindividual capillaries comprising at least one outer wall defining alumen. The outer wall of the capillary can be one or more walls fusedtogether. Similarly, the wall can define a lumen that is cylindrical,square, hexagonal or any other geometric shape so long as the walls forma lumen for retention of a liquid or sample. The capillaries of thecapillary array can be held together in close proximity to form a planarstructure. The capillaries can be bound together, by being fused (e.g.,where the capillaries are made of glass), glued, bonded, or clampedside-by-side. The capillary array can be formed of any number ofindividual capillaries, for example, a range from 100 to 4,000,000capillaries. A capillary array can form a micro titer plate having about100,000 or more individual capillaries bound together.

Arrays, or “Biochips”

Nucleic acids or polypeptides of the invention can be immobilized to orapplied to an array. Arrays can be used to screen for or monitorlibraries of compositions (e.g., small molecules, antibodies, nucleicacids, etc.) for their ability to bind to or modulate the activity of anucleic acid or a polypeptide of the invention. For example, in oneaspect of the invention, a monitored parameter is transcript expressionof an amylase gene. One or more, or, all the transcripts of a cell canbe measured by hybridization of a sample comprising transcripts of thecell, or, nucleic acids representative of or complementary totranscripts of a cell, by hybridization to immobilized nucleic acids onan array, or “biochip.” By using an “array” of nucleic acids on amicrochip, some or all of the transcripts of a cell can besimultaneously quantified. Alternatively, arrays comprising genomicnucleic acid can also be used to determine the genotype of a newlyengineered strain made by the methods of the invention. Polypeptidearrays” can also be used to simultaneously quantify a plurality ofproteins. The present invention can be practiced with any known “array,”also referred to as a “microarray” or “nucleic acid array” or“polypeptide array” or “antibody array” or “biochip,” or variationthereof. Arrays are generically a plurality of “spots” or “targetelements,” each target element comprising a defined amount of one ormore biological molecules, e.g., oligonucleotides, immobilized onto adefined area of a substrate surface for specific binding to a samplemolecule, e.g., mRNA transcripts.

In practicing the methods of the invention, any known array and/ormethod of making and using arrays can be incorporated in whole or inpart, or variations thereof, as described, for example, in U.S. Pat.Nos. 6,277,628; 6,277,489; 6,261,776; 6,258,606; 6,054,270; 6,048,695;6,045,996; 6,022,963; 6,013,440; 5,965,452; 5,959,098; 5,856,174;5,830,645; 5,770,456; 5,632,957; 5,556,752; 5,143,854; 5,807,522;5,800,992; 5,744,305; 5,700,637; 5,556,752; 5,434,049; see also, e.g.,WO 99/51773; WO 99/09217; WO 97/46313; WO 96/17958; see also, e.g.,Johnston (1998) Curr. Biol. 8:R171-R174; Schummer (1997) Biotechniques23:1087-1092; Kern (1997) Biotechniques 23:120-124; Solinas-Toldo (1997)Genes, Chromosomes & Cancer 20:399-407; Bowtell (1999) Nature GeneticsSupp. 21:25-32. See also published U.S. patent applications Nos.20010018642; 20010019827; 20010016322; 20010014449; 20010014448;20010012537; 20010008765.

Antibodies and Antibody-Based Screening Methods

The invention provides isolated or recombinant antibodies thatspecifically bind to an amylase of the invention. These antibodies canbe used to isolate, identify or quantify the amylases of the inventionor related polypeptides. These antibodies can be used to isolate otherpolypeptides within the scope the invention or other related amylases.The antibodies can be designed to bind to an active site of an amylase.Thus, the invention provides methods of inhibiting amylases using theantibodies of the invention.

The antibodies can be used in immunoprecipitation, staining,immunoaffinity columns, and the like. If desired, nucleic acid sequencesencoding for specific antigens can be generated by immunization followedby isolation of polypeptide or nucleic acid, amplification or cloningand immobilization of polypeptide onto an array of the invention.Alternatively, the methods of the invention can be used to modify thestructure of an antibody produced by a cell to be modified, e.g., anantibody's affinity can be increased or decreased. Furthermore, theability to make or modify antibodies can be a phenotype engineered intoa cell by the methods of the invention.

Methods of immunization, producing and isolating antibodies (polyclonaland monoclonal) are known to those of skill in the art and described inthe scientific and patent literature, see, e.g., Coligan, CURRENTPROTOCOLS IN IMMUNOLOGY, Wiley/Greene, N.Y. (1991); Stites (eds.) BASICAND CLINICAL IMMUNOLOGY (7th ed.) Lange Medical Publications, Los Altos,Calif. (“Stites”); Goding, MONOCLONAL ANTIBODIES: PRINCIPLES ANDPRACTICE (2d ed.) Academic Press, New York, N.Y. (1986); Kohler (1975)Nature 256:495; Harlow (1988) ANTIBODIES, A LABORATORY MANUAL, ColdSpring Harbor Publications, New York. Antibodies also can be generatedin vitro, e.g., using recombinant antibody binding site expressing phagedisplay libraries, in addition to the traditional in vivo methods usinganimals. See, e.g., Hoogenboom (1997) Trends Biotechnol. 15:62-70; Katz(1997) Annu. Rev. Biophys. Biomol. Struct. 26:27-45.

Polypeptides or peptides can be used to generate antibodies which bindspecifically to the polypeptides, e.g., the amylases, of the invention.The resulting antibodies may be used in immunoaffinity chromatographyprocedures to isolate or purify the polypeptide or to determine whetherthe polypeptide is present in a biological sample. In such procedures, aprotein preparation, such as an extract, or a biological sample iscontacted with an antibody capable of specifically binding to one of thepolypeptides of the invention.

In immunoaffinity procedures, the antibody is attached to a solidsupport, such as a bead or other column matrix. The protein preparationis placed in contact with the antibody under conditions in which theantibody specifically binds to one of the polypeptides of the invention.After a wash to remove non-specifically bound proteins, the specificallybound polypeptides are eluted.

The ability of proteins in a biological sample to bind to the antibodymay be determined using any of a variety of procedures familiar to thoseskilled in the art. For example, binding may be determined by labelingthe antibody with a detectable label such as a fluorescent agent, anenzymatic label, or a radioisotope. Alternatively, binding of theantibody to the sample may be detected using a secondary antibody havingsuch a detectable label thereon. Particular assays include ELISA assays,sandwich assays, radioimmunoassays, and Western Blots.

Polyclonal antibodies generated against the polypeptides of theinvention can be obtained by direct injection of the polypeptides intoan animal or by administering the polypeptides to a non-human animal.The antibody so obtained will then bind the polypeptide itself. In thismanner, even a sequence encoding only a fragment of the polypeptide canbe used to generate antibodies which may bind to the whole nativepolypeptide. Such antibodies can then be used to isolate the polypeptidefrom cells expressing that polypeptide.

For preparation of monoclonal antibodies, any technique which providesantibodies produced by continuous cell line cultures can be used.Examples include the hybridoma technique, the trioma technique, thehuman B-cell hybridoma technique, and the EBV-hybridoma technique (see,e.g., Cole (1985) in Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, Inc., pp. 77-96).

Techniques described for the production of single chain antibodies (see,e.g., U.S. Pat. No. 4,946,778) can be adapted to produce single chainantibodies to the polypeptides of the invention. Alternatively,transgenic mice may be used to express humanized antibodies to thesepolypeptides or fragments thereof.

Antibodies generated against the polypeptides of the invention may beused in screening for similar polypeptides (e.g., amylases) from otherorganisms and samples. In such techniques, polypeptides from theorganism are contacted with the antibody and those polypeptides whichspecifically bind the antibody are detected. Any of the proceduresdescribed above may be used to detect antibody binding.

Kits

The invention provides kits comprising the compositions, e.g., nucleicacids, expression cassettes, vectors, cells, transgenic seeds or plantsor plant parts, polypeptides (e.g., amylases) and/or antibodies of theinvention. The kits also can contain instructional material teaching themethodologies and industrial uses of the invention, as described herein.

Measuring Metabolic Parameters

The methods of the invention provide whole cell evolution, or whole cellengineering, of a cell to develop a new cell strain having a newphenotype, e.g., a new or modified amylase activity, by modifying thegenetic composition of the cell. The genetic composition can be modifiedby addition to the cell of a nucleic acid of the invention. To detectthe new phenotype, at least one metabolic parameter of a modified cellis monitored in the cell in a “real time” or “on-line” time frame. Inone aspect, a plurality of cells, such as a cell culture, is monitoredin “real time” or “on-line.” In one aspect, a plurality of metabolicparameters is monitored in “real time” or “on-line.” Metabolicparameters can be monitored using the amylases of the invention.

Metabolic flux analysis (MFA) is based on a known biochemistryframework. A linearly independent metabolic matrix is constructed basedon the law of mass conservation and on the pseudo-steady statehypothesis (PSSH) on the intracellular metabolites. In practicing themethods of the invention, metabolic networks are established, includingthe:

-   -   identity of all pathway substrates, products and intermediary        metabolites    -   identity of all the chemical reactions interconverting the        pathway metabolites, the stoichiometry of the pathway reactions,    -   identity of all the enzymes catalyzing the reactions, the enzyme        reaction kinetics,    -   the regulatory interactions between pathway components, e.g.        allosteric interactions, enzyme-enzyme interactions etc,    -   intracellular compartmentalization of enzymes or any other        supramolecular organization of the enzymes, and,    -   the presence of any concentration gradients of metabolites,        enzymes or effector molecules or diffusion barriers to their        movement.

Once the metabolic network for a given strain is built, mathematicpresentation by matrix notion can be introduced to estimate theintracellular metabolic fluxes if the on-line metabolome data isavailable. Metabolic phenotype relies on the changes of the wholemetabolic network within a cell. Metabolic phenotype relies on thechange of pathway utilization with respect to environmental conditions,genetic regulation, developmental state and the genotype, etc. In oneaspect of the methods of the invention, after the on-line MFAcalculation, the dynamic behavior of the cells, their phenotype andother properties are analyzed by investigating the pathway utilization.For example, if the glucose supply is increased and the oxygen decreasedduring the yeast fermentation, the utilization of respiratory pathwayswill be reduced and/or stopped, and the utilization of the fermentativepathways will dominate. Control of physiological state of cell cultureswill become possible after the pathway analysis. The methods of theinvention can help determine how to manipulate the fermentation bydetermining how to change the substrate supply, temperature, use ofinducers, etc. to control the physiological state of cells to move alongdesirable direction. In practicing the methods of the invention, the MFAresults can also be compared with transcriptome and proteome data todesign experiments and protocols for metabolic engineering or geneshuffling, etc.

In practicing the methods of the invention, any modified or newphenotype can be conferred and detected, including new or improvedcharacteristics in the cell. Any aspect of metabolism or growth can bemonitored.

Monitoring Expression of an mRNA Transcript

In one aspect of the invention, the engineered phenotype comprisesincreasing or decreasing the expression of an mRNA transcript (e.g., anamylase message) or generating new (e.g., amylase) transcripts in acell. This increased or decreased expression can be traced by testingfor the presence of an amylase of the invention or by amylase activityassays. mRNA transcripts, or messages, also can be detected andquantified by any method known in the art, including, e.g., Northernblots, quantitative amplification reactions, hybridization to arrays,and the like. Quantitative amplification reactions include, e.g.,quantitative PCR, including, e.g., quantitative reverse transcriptionpolymerase chain reaction, or RT-PCR; quantitative real time RT-PCR, or“real-time kinetic RT-PCR” (see, e.g., Kreuzer (2001) Br. J. Haematol.114:313-318; Xia (2001) Transplantation 72:907-914). In one aspect ofthe invention, the engineered phenotype is generated by knocking outexpression of a homologous gene. The gene's coding sequence or one ormore transcriptional control elements can be knocked out, e.g.,promoters or enhancers. Thus, the expression of a transcript can becompletely ablated or only decreased.

In one aspect of the invention, the engineered phenotype comprisesincreasing the expression of a homologous gene. This can be effected byknocking out of a negative control element, including a transcriptionalregulatory element acting in cis- or trans-, or, mutagenizing a positivecontrol element. One or more, or, all the transcripts of a cell can bemeasured by hybridization of a sample comprising transcripts of thecell, or, nucleic acids representative of or complementary totranscripts of a cell, by hybridization to immobilized nucleic acids onan array.

Monitoring Expression of a Polypeptides, Peptides and Amino Acids

In one aspect of the invention, the engineered phenotype comprisesincreasing or decreasing the expression of a polypeptide (e.g., anamylase) or generating new polypeptides in a cell. This increased ordecreased expression can be traced by determining the amount of amylasepresent or by amylase activity assays. Polypeptides, peptides and aminoacids also can be detected and quantified by any method known in theart, including, e.g., nuclear magnetic resonance (NMR),spectrophotometry, radiography (protein radiolabeling), electrophoresis,capillary electrophoresis, high performance liquid chromatography(HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography,various immunological methods, e.g. immunoprecipitation,immunodiffusion, immuno-electrophoresis, radioimmunoassays (RIAs),enzyme-linked immunosorbent assays (ELISAs), immuno-fluorescent assays,gel electrophoresis (e.g., SDS-PAGE), staining with antibodies,fluorescent activated cell sorter (FACS), pyrolysis mass spectrometry,Fourier-Transform Infrared Spectrometry, Raman spectrometry, GC-MS, andLC-Electrospray and cap-LC-tandem-electrospray mass spectrometries, andthe like. Novel bioactivities can also be screened using methods, orvariations thereof, described in U.S. Pat. No. 6,057,103. Furthermore,as discussed below in detail, one or more, or, all the polypeptides of acell can be measured using a protein array.

Industrial Applications

Detergent Compositions

The invention provides detergent compositions comprising one or morepolypeptides of the invention, and methods of making and using thesecompositions. The invention incorporates all methods of making and usingdetergent compositions, see, e.g., U.S. Pat. Nos. 6,413,928; 6,399,561;6,365,561; 6,380,147. The detergent compositions can be a one and twopart aqueous composition, a non-aqueous liquid composition, a castsolid, a granular form, a particulate form, a compressed tablet, a geland/or a paste and a slurry form. The invention also provides methodscapable of a rapid removal of gross food soils, films of food residueand other minor food compositions using these detergent compositions.Amylases of the invention can facilitate the removal of starchy stainsby means of catalytic hydrolysis of the starch polysaccharide. Amylasesof the invention can be used in dishwashing detergents in textilelaundering detergents.

The actual active enzyme content depends upon the method of manufactureof a detergent composition and is not critical, assuming the detergentsolution has the desired enzymatic activity. In one aspect, the amountof amylase present in the final solution ranges from about 0.001 mg to0.5 mg per gram of the detergent composition. The particular enzymechosen for use in the process and products of this invention dependsupon the conditions of final utility, including the physical productform, use pH, use temperature, and soil types to be degraded or altered.The enzyme can be chosen to provide optimum activity and stability forany given set of utility conditions. In one aspect, the polypeptides ofthe present invention are active in the pH ranges of from about 4 toabout 12 and in the temperature range of from about 20° C. to about 95°C. The detergents of the invention can comprise cationic, semi-polarnonionic or zwitterionic surfactants; or, mixtures thereof.

Amylases of the present invention can be formulated into powdered andliquid detergents having pH between 4.0 and 12.0 at levels of about 0.01to about 5% (preferably 0.1% to 0.5%) by weight. These detergentcompositions can also include other enzymes such as known proteases,cellulases, lipases or endoglycosidases, as well as builders andstabilizers. The addition of amylases of the invention to conventionalcleaning compositions does not create any special use limitation. Inother words, any temperature and pH suitable for the detergent is alsosuitable for the present compositions as long as the pH is within theabove range, and the temperature is below the described enzyme'sdenaturing temperature. In addition, the polypeptides of the inventioncan be used in a cleaning composition without detergents, again eitheralone or in combination with builders and stabilizers.

The present invention provides cleaning compositions including detergentcompositions for cleaning hard surfaces, detergent compositions forcleaning fabrics, dishwashing compositions, oral cleaning compositions,denture cleaning compositions, and contact lens cleaning solutions.

In one aspect, the invention provides a method for washing an objectcomprising contacting the object with a polypeptide of the inventionunder conditions sufficient for washing. A polypeptide of the inventionmay be included as a detergent additive. The detergent composition ofthe invention may, for example, be formulated as a hand or machinelaundry detergent composition comprising a polypeptide of the invention.A laundry additive suitable for pre-treatment of stained fabrics cancomprise a polypeptide of the invention. A fabric softener compositioncan comprise a polypeptide of the invention. Alternatively, apolypeptide of the invention can be formulated as a detergentcomposition for use in general household hard surface cleaningoperations. In alternative aspects, detergent additives and detergentcompositions of the invention may comprise one or more other enzymessuch as a protease, a lipase, a cutinase, another amylase, acarbohydrase, a cellulase, a pectinase, a mannanase, an arabinase, agalactanase, a xylanase, an oxidase, e.g., a lactase, and/or aperoxidase. The properties of the enzyme(s) of the invention are chosento be compatible with the selected detergent (i.e. pH-optimum,compatibility with other enzymatic and non-enzymatic ingredients, etc.)and the enzyme(s) is present in effective amounts. In one aspect,amylase enzymes of the invention are used to remove malodorous materialsfrom fabrics. Various detergent compositions and methods for making themthat can be used in practicing the invention are described in, e.g.,U.S. Pat. Nos. 6,333,301; 6,329,333; 6,326,341; 6,297,038; 6,309,871;6,204,232; 6,197,070; 5,856,164.

Treating Fabrics

The invention provides methods of treating fabrics using one or morepolypeptides of the invention. The polypeptides of the invention can beused in any fabric-treating method, which are well known in the art,see, e.g., U.S. Pat. No. 6,077,316. For example, in one aspect, the feeland appearance of a fabric is improved by a method comprising contactingthe fabric with an amylase of the invention in a solution. In oneaspect, the fabric is treated with the solution under pressure.

In one aspect, the enzymes of the invention are applied during or afterthe weaving of textiles, or during the desizing stage, or one or moreadditional fabric processing steps. During the weaving of textiles, thethreads are exposed to considerable mechanical strain. Prior to weavingon mechanical looms, warp yarns are often coated with sizing starch orstarch derivatives in order to increase their tensile strength and toprevent breaking. The enzymes of the invention can be applied to removethese sizing starch or starch derivatives. After the textiles have beenwoven, a fabric can proceed to a desizing stage. This can be followed byone or more additional fabric processing steps. Desizing is the act ofremoving size from textiles. After weaving, the size coating must beremoved before further processing the fabric in order to ensure ahomogeneous and wash-proof result. The invention provides a method ofdesizing comprising enzymatic hydrolysis of the size by the action of anenzyme of the invention.

The enzymes of the invention can be used to desize fabrics, includingcotton-containing fabrics, as detergent additives, e.g., in aqueouscompositions. The invention provides methods for producing a stonewashedlook on indigo-dyed denim fabric and garments. For the manufacture ofclothes, the fabric can be cut and sewn into clothes or garments, whichis afterwards finished. In particular, for the manufacture of denimjeans, different enzymatic finishing methods have been developed. Thefinishing of denim garment normally is initiated with an enzymaticdesizing step, during which garments are subjected to the action ofamylolytic enzymes in order to provide softness to the fabric and makethe cotton more accessible to the subsequent enzymatic finishing steps.The invention provides methods of finishing denim garments (e.g., a“bio-stoning process”), enzymatic desizing and providing softness tofabrics using the amylases of the invention. The invention providesmethods for quickly softening denim garments in a desizing and/orfinishing process.

Foods and Food Processing

The enzymes of the invention have numerous applications in foodprocessing industry. The amylases of the invention are used in starch tofructose processing. Starch to fructose processing can consist of foursteps: liquefaction of granular starch, saccharification of theliquefied starch into dextrose, purification, and isomerization tofructose.

The invention provides methods of starch liquefaction using the enzymesof the invention. Concentrated suspensions of starch polymer granulesare converted into a solution of soluble shorter chain length dextrinsof low viscosity. This step is useful for convenient handling withstandard equipment and for efficient conversion to glucose or 10³ othersugars. In one aspect, the granular starch is liquefied by gelatinizingthe granules by raising the temperature of the granular starch to overabout 72° C. The heating process instantaneously disrupts the insolublestarch granules to produce a water soluble starch solution. Thesolubilized starch solution can then be liquefied by an amylase of theinvention. Thus, the invention provides enzymatic starch liquefactionprocesses using an amylase of the invention.

An exemplary enzymatic liquefaction process involves adjusting the pH ofa granular starch slurry to between 6.0 and 6.5 and the addition ofcalcium hydroxide, sodium hydroxide or sodium carbonate. In one aspect,calcium hydroxide is added. This provides calcium ions to stabilize theglucoamylase of the invention against inactivation. In one aspect, uponaddition of amylase, the suspension is pumped through a steam jet toinstantaneously raise the temperature to between 80°-115° C. In oneaspect, the starch is immediately gelatinized and, due to the presenceof amylase, depolymerized through random hydrolysis of α-1,4-glycosidicbonds by amylase to a fluid mass. The fluid mass can be easily pumped.

The invention provides various enzymatic starch liquefaction processesusing an amylase of the invention. In one aspect of the liquefactionprocess of the invention, an amylase is added to the starch suspensionand the suspension is held at a temperature of between about 80°-100° C.to partially hydrolyze the starch granules. In one aspect, the partiallyhydrolyzed starch suspension is pumped through a jet at temperatures inexcess of about 105° C. to thoroughly gelatinize any remaining granularstructure. In one aspect, after cooling the gelatinized starch, a secondaddition of amylase is made to further hydrolyze the starch.

The invention provides enzymatic dry milling processes using an amylaseof the invention. In dry milling, whole grain is ground and combinedwith water. The germ is optionally removed by flotation separation orequivalent techniques. The resulting mixture, which contains starch,fiber, protein and other components of the grain, is liquefied usingamylase. In one aspect, enzymatic liquefaction is done at lowertemperatures than the starch liquification processes discussed above. Inone aspect, after gelatinization the starch solution is held at anelevated temperature in the presence of amylase until a DE of 10-20 isachieved. In one aspect, this is a period of about 1-3 hours. Dextroseequivalent (DE) is the industry standard for measuring the concentrationof total reducing sugars, calculated as D-glucose on a dry weight basis.Unhydrolyzed granular starch has a DE of virtually zero, whereas the DEof D-glucose is defined as 100.

The invention provides wet milling processes, e.g., corn wet milling,using an amylase of the invention. Corn wet milling is a process whichproduces corn oil, gluten meal, gluten feed and starch. Thus, theinvention provides methods of making corn oil, gluten meal, gluten feedand starch using an enzyme of the invention. In one aspect, analkaline-amylase of the invention is used in the liquefaction of starch.In one aspect, glucoamylase is used in saccharification to produceglucose.

In one aspect, corn (a kernel that consists of a outer seed coat(fiber), starch, a combination of starch and glucose and the innergerm), is subjected to a four step process, which results in theproduction of starch. In one aspect, the corn is steeped, de-germed,de-fibered, and the gluten is separated. In a steeping process thesolubles are taken out. The product remaining after removal of thesolubles is de-germed, resulting in production of corn oil andproduction of an oil cake, which is added to the solubles from thesteeping step. The remaining product is de-fibered and the fiber solidsare added to the oil cake/solubles mixture. This mixture of fibersolids, oil cake and solubles forms a gluten feed. After de-fibering,the remaining product is subjected to gluten separation. This separationresults in a gluten meal and starch. The starch is then subjected toliquefaction and saccharification using polypeptides of the invention toproduce glucose.

The invention provides anti-staling processes (e.g., of baked productssuch as bread) using an amylase of the invention. The invention providesmethods to slow the increase of the firmness of the crumb (of the bakedproduct) and a decrease of the elasticity of the crumb using an amylaseof the invention. Staling of baked products (such as bread) is moreserious as time passes between the moment of preparation of the breadproduct and the moment of consumption. The term staling is used todescribe changes undesirable to the consumer in the properties of thebread product after leaving the oven, such as an increase of thefirmness of the crumb, a decrease of the elasticity of the crumb, andchanges in the crust, which becomes tough and leathery. The firmness ofthe bread crumb increases further during storage up to a level, which isconsidered as negative. Amylases of the invention are used to retardstaling of the bread as described e.g., in U.S. Pat. Nos. 6,197,352;2,615,810 and 3,026,205; Silberstein (1964) Baker's Digest 38:66-72.

In one aspect, an enzyme of the invention is used to retard the stalingof baked products while not hydrolyzing starch into the brancheddextrins. Branched dextrins are formed by cleaving off the branchedchains of the dextrins generated by α-amylase hydrolysis which cannot bedegraded further by the α-amylase. This can produce a gummy crumb in theresulting bread. Accordingly, the invention provides a process forretarding the staling of baked products (e.g., leavened baked products)comprising adding an enzyme of the invention comprising exoamylaseactivity to a flour or a dough used for producing a baked product.Exoamylases of the invention can have glucoamylase, β-amylase (whichreleases maltose in the beta-configuration) and/or maltogenic amylaseactivity.

The invention also provides a process for preparing a dough or a bakedproduct prepared from the dough which comprises adding an amylase of theinvention to the dough in an amount which is effective to retard thestaling of the bread. The invention also provides a dough comprisingsaid amylase and a premix comprising flour together with said amylase.Finally, the invention provides an enzymatic baking additive, whichcontains said amylase.

The invention also provides a high yield process for producing highquality corn fiber gum by treatment of corn fiber with an enzyme of theinvention followed by hydrogen peroxide treatment to obtain an extractof milled corn fiber. See, e.g., U.S. Pat. No. 6,147,206.

Animal Feeds and Additives

The invention provides methods for treating animal feeds and additivesusing amylase enzymes of the invention. The invention provides animalfeeds and additives comprising amylases of the invention. In one aspect,treating animal feeds and additives using amylase enzymes of theinvention can help in the availability of starch in the animal feed oradditive. This can result in release of readily digestible and easilyabsorbed sugars.

Use of an amylase of the invention can increase the digestive capacityof animals and birds. Use of an amylase of the invention can ensureavailability of an adequate nutrient supply for better growth andperformance. In one aspect, the enzymes of the invention can be added asfeed additives for animals. In another aspect, the animal feed can betreated with amylases prior to animal consumption. In another aspect,the amylases may be supplied by expressing the enzymes directly intransgenic feed crops (as, e.g., transgenic plants, seeds and the like),such as corn. As discussed above, the invention provides transgenicplants, plant parts and plant cells comprising a nucleic acid sequenceencoding a polypeptide of the invention. In one aspect, the nucleic acidis expressed such that the amylase is produced in recoverablequantities. The amylase can be recovered from any plant or plant part.Alternatively, the plant or plant part containing the recombinantpolypeptide can be used as such for improving the quality of a food orfeed, e.g., improving nutritional value, palatability, and rheologicalproperties, or to destroy an antinutritive factor.

Paper or Pulp Treatment

The enzymes of the invention can be in paper or pulp treatment or paperdeinking. For example, in one aspect, the invention provides a papertreatment process using amylases of the invention. In one aspect, theenzymes of the invention can be used to modify starch in the paperthereby converting it into a liquefied form. In another aspect, papercomponents of recycled photocopied paper during chemical and enzymaticdeinking processes. In one aspect, amylases of the invention can be usedin combination with cellulases. The paper can be treated by thefollowing three processes: 1) disintegration in the presence of anenzyme of the invention, 2) disintegration with a deinking chemical andan enzyme of the invention, and/or 3) disintegration after soaking withan enzyme of the invention. The recycled paper treated with amylase canhave a higher brightness due to removal of toner particles as comparedto the paper treated with just cellulase. While the invention is notlimited by any particular mechanism, the effect of an amylase of theinvention may be due to its behavior as surface-active agents in pulpsuspension.

The invention provides methods of treating paper and paper pulp usingone or more polypeptides of the invention. The polypeptides of theinvention can be used in any paper- or pulp-treating method, which arewell known in the art, see, e.g., U.S. Pat. Nos. 6,241,849; 6,066,233;5,582,681. For example, in one aspect, the invention provides a methodfor deinking and decolorizing a printed paper containing a dye,comprising pulping a printed paper to obtain a pulp slurry, anddislodging an ink from the pulp slurry in the presence of an enzyme ofthe invention (other enzymes can also be added). In another aspect, theinvention provides a method for enhancing the freeness of pulp, e.g.,pulp made from secondary fiber, by adding an enzymatic mixturecomprising an enzyme of the invention (can also include other enzymes,e.g., pectinase enzymes) to the pulp and treating under conditions tocause a reaction to produce an enzymatically treated pulp. The freenessof the enzymatically treated pulp is increased from the initial freenessof the secondary fiber pulp without a loss in brightness.

Repulping: Treatment of Lignocellulosic Materials

The invention also provides a method for the treatment oflignocellulosic fibers, wherein the fibers are treated with apolypeptide of the invention, in an amount which is efficient forimproving the fiber properties. The amylases of the invention may alsobe used in the production of lignocellulosic materials such as pulp,paper and cardboard, from starch reinforced waste paper and cardboard,especially where repulping occurs at pH above 7 and where amylases canfacilitate the disintegration of the waste material through degradationof the reinforcing starch. The amylases of the invention can be usefulin a process for producing a papermaking pulp from starch-coated printedpaper. The process may be performed as described in, e.g., WO 95/14807.

An exemplary process comprises disintegrating the paper to produce apulp, treating with a starch-degrading enzyme before, during or afterthe disintegrating, and separating ink particles from the pulp afterdisintegrating and enzyme treatment. See also U.S. Pat. No. 6,309,871and other US patents cited herein. Thus, the invention includes a methodfor enzymatic deinking of recycled paper pulp, wherein the polypeptideis applied in an amount which is efficient for effective de-inking ofthe fiber surface.

Waste Treatment

The enzymes of the invention can be used in a variety of otherindustrial applications, e.g., in waste treatment. For example, in oneaspect, the invention provides a solid waste digestion process usingenzymes of the invention. The methods can comprise reducing the mass andvolume of substantially untreated solid waste. Solid waste can betreated with an enzymatic digestive process in the presence of anenzymatic solution (including an enzyme of the invention) at acontrolled temperature. This results in a reaction without appreciablebacterial fermentation from added microorganisms. The solid waste isconverted into a liquefied waste and any residual solid waste. Theresulting liquefied waste can be separated from said any residualsolidified waste. See e.g., U.S. Pat. No. 5,709,796.

Oral Care Products

The invention provides oral care product comprising an amylase of theinvention. Exemplary oral care products include toothpastes, dentalcreams, gels or tooth powders, odontics, mouth washes, pre- or postbrushing rinse formulations, chewing gums, lozenges, or candy. See,e.g., U.S. Pat. No. 6,264,925.

Brewing and Fermenting

The invention provides methods of brewing (e.g., fermenting) beercomprising an amylase of the invention. In one exemplary process,starch-containing raw materials are disintegrated and processed to forma malt. An amylase of the invention is used at any point in thefermentation process. For example, amylases of the invention can be usedin the processing of barley malt. The major raw material of beer brewingis barley malt. This can be a three stage process. First, the barleygrain can be steeped to increase water content, e.g., to around about40%. Second, the grain can be germinated by incubation at 15-25° C. for3 to 6 days when enzyme synthesis is stimulated under the control ofgibberellins. During this time amylase levels rise significantly. In oneaspect, amylases of the invention are added at this (or any other) stageof the process. The action of the amylase results in an increase infermentable reducing sugars. This can be expressed as the diastaticpower, DP, which can rise from around 80 to 190 in 5 days at 12° C.

Amylases of the invention can be used in any beer producing process, asdescribed, e.g., in U.S. Pat. Nos. 5,762,991; 5,536,650; 5,405,624;5,021,246; 4,788,066.

Other Industrial Applications

The invention also includes a method of increasing the flow ofproduction fluids from a subterranean formation by removing a viscous,starch-containing, damaging fluid formed during production operationsand found within the subterranean formation which surrounds a completedwell bore comprising allowing production fluids to flow from the wellbore; reducing the flow of production fluids from the formation belowexpected flow rates; formulating an enzyme treatment by blendingtogether an aqueous fluid and a polypeptide of the invention; pumpingthe enzyme treatment to a desired location within the well bore;allowing the enzyme treatment to degrade the viscous, starch-containing,damaging fluid, whereby the fluid can be removed from the subterraneanformation to the well surface; and wherein the enzyme treatment iseffective to attack the alpha glucosidic linkages in thestarch-containing fluid.

In summary, the invention provides enzymes and processes for hydrolyzingliquid (liquefied) and granular starch. Such starch can be derived fromany source, e.g., corn, wheat, milo, sorghum, rye or bulgher. Theinvention applies to any grain starch source which is useful inliquefaction, e.g., any other grain or vegetable source known to producestarch suitable for liquefaction. The methods of the invention compriseliquefying starch from any natural material, such as rice, germinatedrice, corn, barley, milo, wheat, legumes and sweet potato. Theliquefying process can substantially hydrolyze the starch to produce asyrup. The temperature range of the liquefaction can be any liquefactiontemperature which is known to be effective in liquefying starch. Forexample, the temperature of the starch can be between about 80° C. toabout 115° C., between about 100° C. to about 110° C., and from about105° C. to about 108° C.

In one aspect, the invention includes a method for liquefying a starchcontaining composition comprising contacting the starch with apolypeptide of the invention (e.g., a purified polypeptide selected frompolypeptides having an amino acid sequence selected from the groupconsisting of: SEQ ID NO:2; variants having at least about 50% homologyto at least one of SEQ ID NO:2, over a region of at least about 100residues, as determined by analysis with a sequence comparison algorithmor by visual inspection; sequences complementary to SEQ ID NO:2; andsequences complementary to variants having at least about 50% homologyto SEQ ID NO:2 over a region of at least about 100 residues, asdetermined by analysis with a sequence comparison algorithm or by visualinspection; and polypeptides having at least 10 consecutive amino acidsof a polypeptide having a sequence selected from the group consisting ofSEQ ID NO:2). In one aspect, the polypeptide is set forth in SEQ IDNO:2. The starch may be from a material selected from rice, germinatedrice, corn, barley, wheat, legumes and sweet potato. A glucose syrupproduced by the method of the invention is included herein and isdescribed in the examples. Such a syrup can be a maltose syrup, aglucose syrup, or a combination thereof. In particular, the syrupsproduced using the amylases of the invention there is a higher level ofDP2 fraction and a higher level of DP3 (maltotriose and/or panose) andless of the greater than DP7 fragments as compared to the syrupsproduced by commercial enzymes. This is consistent with the liquefactionprofile since less of the large fragments are in the invention liquefiedsyrups.

The invention also provides a method for removing starch containingstains from a material comprising contacting the material with apolypeptide of the invention. In one aspect, the invention provides amethod for washing an object comprising contacting the object with apolypeptide of the invention under conditions sufficient for washing. Apolypeptide of the invention may be included as a detergent additive forexample. The invention also includes a method for textile desizingcomprising contacting the textile with a polypeptide of the inventionunder conditions sufficient for desizing.

The invention also provides a method of reducing the staling of bakeryproducts comprising addition of a polypeptide of the invention to thebakery product, prior to baking.

The invention also provides a method for the treatment oflignocellulosic fibers, wherein the fibers are treated with apolypeptide of the invention, in an amount which is efficient forimproving the fiber properties. The invention includes a method forenzymatic deinking of recycled paper pulp, wherein the polypeptide isapplied in an amount which is efficient for effective deinking of thefiber surface.

Any of the methods described herein include the possibility of theaddition of a second alpha amylase or a beta amylase or a combinationthereof. Commercial amylases or other enzymes suitable for use incombination with an enzyme of the invention are known to those of skillin the art.

The invention also includes a method of increasing the flow ofproduction fluids from a subterranean formation by removing a viscous,starch-containing, damaging fluid formed during production operationsand found within the subterranean formation which surrounds a completedwell bore comprising allowing production fluids to flow from the wellbore; reducing the flow of production fluids from the formation belowexpected flow rates; formulating an enzyme treatment by blendingtogether an aqueous fluid and a polypeptide of the invention; pumpingthe enzyme treatment to a desired location within the well bore;allowing the enzyme treatment to degrade the viscous, starch-containing,damaging fluid, whereby the fluid can be removed from the subterraneanformation to the well surface; and wherein the enzyme treatment iseffective to attack the alpha glucosidic linkages in thestarch-containing fluid.

The present invention exploits the unique catalytic properties ofenzymes. Whereas the use of biocatalysts (i.e., purified or crudeenzymes, non-living or living cells) in chemical transformationsnormally requires the identification of a particular biocatalyst thatreacts with a specific starting compound, the present invention usesselected biocatalysts and reaction conditions that are specific forfunctional groups that are present in many starting compounds. Eachbiocatalyst is specific for one functional group, or several relatedfunctional groups, and can react with many starting compounds containingthis functional group. The biocatalytic reactions produce a populationof derivatives from a single starting compound. These derivatives can besubjected to another round of biocatalytic reactions to produce a secondpopulation of derivative compounds. Thousands of variations of theoriginal compound can be produced with each iteration of biocatalyticderivatization.

Enzymes react at specific sites of a starting compound without affectingthe rest of the molecule, a process which is very difficult to achieveusing traditional chemical methods. This high degree of biocatalyticspecificity provides the means to identify a single active compoundwithin the library. The library is characterized by the series ofbiocatalytic reactions used to produce it, a so-called “biosynthetichistory”. Screening the library for biological activities and tracingthe biosynthetic history identifies the specific reaction sequenceproducing the active compound. The reaction sequence is repeated and thestructure of the synthesized compound determined. This mode ofidentification, unlike other synthesis and screening approaches, doesnot require immobilization technologies, and compounds can besynthesized and tested free in solution using virtually any type ofscreening assay. It is important to note, that the high degree ofspecificity of enzyme reactions on functional groups allows for the“tracking” of specific enzymatic reactions that make up thebiocatalytically produced library.

Many of the procedural steps are performed using robotic automationenabling the execution of many thousands of biocatalytic reactions andscreening assays per day as well as ensuring a high level of accuracyand reproducibility. As a result, a library of derivative compounds canbe produced in a matter of weeks which would take years to produce usingcurrent chemical methods. (For further teachings on modification ofmolecules, including small molecules, see, e.g., PCT/US94/09174).

The invention will be further described with reference to the followingexamples; however, it is to be understood that the invention is notlimited to such examples.

EXAMPLES Example 1: Identification and Characterization of Thermostableα-Amylases

The following example describes an exemplary method for determining if apolypeptide is within the scope of the invention. This example describesthe identification of novel amylases. A screening program can be carriedout under neutral and/or low pH conditions. DNA libraries generated fromlow pH samples can be targeted for discovery.

Biochemical Studies

Biochemical analysis of genomic clones can show if any have pH optima ofless than pH 6. Lysates of these genomic clones can be tested forthermal tolerance by incubation at 70° C., 80° C., 90° C. or 100° C. for10 minutes and measurement of residual activity at pH 4.5. Those clonesretaining >50% activity after heat treatment at 80° C. can be chosen forfurther analysis. These clones can be incubated at 90° C. for 10 minutesat pH 6.0 and 4.5 and tested for residual activity at pH 4.5.

Thermal activity of the clones with residual activity after heattreatment at 90° C. at pH 4.5 can be measured at room temperature, 70°C. and 90° C. at pH 4.5.

Example 2: Thermostable Amylases Active at Alkaline pH

The following example describes an exemplary method for determining if apolypeptide is within the scope of the invention, e.g., is athermostable amylase.

Biochemical Studies

Soluble, active protein can be successfully purified to homogeneity andmeasured at pH 8 and pH 10 (40° C. and 50° C.) using 2% starch inbuffer.

Stability

Activity can be measured at pH 8 and 50° C. after a 30 minute incubationat 50° C. in the presence of various components of an ADW formulation;pH 8, pH 10.8, ADW solution (with bleach) and ADW solution (withoutbleach). The measured activity after the incubation can be expressed asa percentage of the original activity.

Wash Tests

Wash tests using starch coated slides can be carried out to gauge theperformance of each of the purified enzymes as compared to thecommercial amylase. The spaghetti starch coated slides can be preparedaccording to manufacturer's protocol. Two pre-weighed starch coatedslides can be placed back to back in a 50 mL conical tube and 25 mL ofADW solution, +/− enzyme were added per tube. The tubes can be incubatedfor 20 minutes at 50° C. with gentle rotation on a vertical carousel.Following the incubation period, the slides are immediately rinsed inwater and oven dried overnight. Trials can be run in duplicate and acommercial enzyme can be run as a positive control.

Example 3: Amylase Activity Assay: BCA Reducing Ends Assay

The following example describes an exemplary method for determining if apolypeptide is within the scope of the invention, for example, by a BCAreducing ends assay. Amylase activity of clones of interest wasdetermined using the following methodology.

1. Prepare 2 substrate solutions, as follows:

-   -   a) 2% soluble starch (potato) pH 8 solution by dissolving 2 gm        potato starch in 100 ml 100 mM sodium phosphate pH 8).    -   b) 2% soluble starch (potato) pH 10 solution by dissolving 2 gm        potato starch in 100 ml 100 mM sodium carbonate.

Heat both solutions in a boiling water bath, while mixing, for 30-40minutes until starch dissolves.

2. Prepare Solution A from 64 mg/ml sodium carbonate monohydrate, 24mg/ml sodium bicarbonate and 1.95 mg/ml BCA(4,4′-dicarboxy-2,2′-biquinoline disodium salt (Sigma Chemical cat #D-8284). Added above to dH2O.

3. Prepare solution B by combining 1.24 mg/ml cupric sulfatepentahydrate and 1.26 mg/ml L-serine. Add mixture to dH2O.

4. Prepare a working reagent of a 1:1 ration of solutions A and B.

5. Prepare a Maltose standard solution of 10 mM Maltose in dH2O, wherethe 10 mM maltose is combined in 2% soluble starch at desired pH to afinal concentration of 0, 100, 200, 300, 400, 600 μM. The standard curvewill be generated for each set of time-points. Since the curve isdetermined by adding 10 ul of the standards to the working reagent itworks out to 0, 1, 2, 3, 4, 6 nmole maltose. See FIG. 5 for an examplestandard curve.

6. Aliquot 1 ml of substrate solution into microcentrifuge tubes,equilibrate to desired temperature (5 min) in heat block or heated waterbath. Add 50 ul of enzyme solution to the inside of the tube lid.

7. While solution is equilibrating mix 5 ml of both solution A & B.Aliquot 100 ul to 96 well PCR plate. Set plate on ice.

8. After 5 minute temperature equilibration, close lid on tubes, invertand vortex 3 times. Immediately aliquot 10 ul into plate as t=0 (zerotime point). Leave enzyme mixture in heat block and aliquot 10 ul ateach desired time point (e.g. 0, 5, 10, 15, 20, 30 minutes).

9. Ensure that 12 wells are left empty (only working reagent aliquotted)for the addition of 10 ul of standards, for the standard curve.

10. When all time points are collected and standards are added, coverplate and heated to 80° C. for 35 min. Cool plate on ice for 10 min. Add100 ul H₂O to all wells. Mix and aliquot 100 ul into flat bottomed96-well plate and read absorbance at 560 nm.

11. Zero each sample's time points against its own t=0 (subtract theaverage t=0 A560 value from other average A560 values). Convert theA560_((experimental)) to umole (Divide A560_((experimental)) by theslope of the standard curve (A560/umole). Generate a slope of the timepoints and the umole (in umole/min), multiply by 100 (as the umole valueonly accounts for the 10 ul used in the assay, not the amount made inthe 1 ml r×n). To get the specific activity divide the slope (inumole/min) by the mg of protein. All points should be done at a minimumin duplicate with three being best.

-   -   Activity=0.008646 umole/min    -   Divide protein concentration (mg/ml) by any dilution to get mg        used in assay.    -   Divide the above slope by mg used in assay to get specific        activity    -   Specific Activity=24.93 umole/min/mg

See for example, Dominic W. S. Wong, Sarah B. Batt, and George H.Robertson (2000) J. Agric. Food Chem. 48:4540-4543; Jeffrey D. Fox andJohn F. Robyt, (1991) Anal. Biochem. 195, 93-96.

Example 4: Screening for α-Amylase Activity

The following example describes an exemplary method for determining if apolypeptide is within the scope of the invention. Amylase activity ofclones can be assessed by a number of methods known in the art. Thefollowing is the general methodology that was used in the presentinvention. The number of plaques screened, per plate, should beapproximately 10,000 pfu's. For each DNA library: at least 50,000plaques per isolated library and 200,000 plaques per non-isolatedlibrary should be screened depending upon the pfu titer for the λ, ZapExpress amplified lysate.

Titer Determination of Lambda Library

-   1) μL of Lambda Zap Express amplified library stock added to 600    μL E. coli MRF′ cells (OD600=1.0). To dilute MRF′ stock, 10 mM MgSO₄    is used.-   2) Incubate at 37° C. for 15 minutes.-   3) Transfer suspension to 5-6 mL of NZY top agar at 50° C. and    gently mix.-   4) Immediately pour agar solution onto large (150 mm) NZY media    plate.-   5) Allow top agar to solidify completely (approximately 30 minutes),    then invert plate.-   6) Incubate the plate at 39° C. for 8-12 hours.-   7) Number of plaques is approximated. Phage titer determined to give    10,000 pfu/plate. Dilute an aliquot of Library phage with SM buffer    if needed.

Substrate Screening

-   1) Lambda Zap Express (50,000 pfu) from amplified library added to    600 μL of E. coli MRF′ cells (OD600=1.0). For non-environment    libraries, prepare 4 tubes (50,000 pfu per tube).-   2) Incubate at 37° C. for 15 minutes.-   3) While phage/cell suspension are incubating, 1.0 mL of red starch    substrate (1.2% w/v) is added to 6.0 mL NZY top agar at 50° C. and    mixed thoroughly. Keep solution at 50° C. until needed.-   4) Transfer ⅕ (10,000 pfu) of the cell suspension to substrate/top    agar solution and gently mixed.-   5) Solution is immediately poured onto large (150 mm) NZY media    plate.-   6) Allow top agar to solidify completely (approximately 30 minutes),    then invert plate.-   7) Repeat procedures 4-6 4 times for the rest of the cell suspension    (⅕ of the suspension each time).-   8) Incubate plates at 39° C. for 8-12 hours.-   9) Plate observed for clearing zones (halos) around plaques.-   10) Plaques with halos are cored out of agar and transferred to a    sterile micro tube. A large bore 200 μL pipette tip works well to    remove (core) the agar plug containing the desired plaque.-   11) Phages are re-suspended in 500 μL SM buffer. 20 μL Chloroform is    added to inhibit any further cell growth.-   12) Pure phage suspension is incubated at room temperature for 4    hours or overnight before next step.

Isolation of Pure Clones

-   1) 10 μL of re-suspended phage suspension is added to 500 μL of E.    coli MRF′ cells (OD600=1.0).-   2) Incubate at 37° C. for 15 minutes.-   3) While phage/cell suspension is incubating, 1 mL of red starch    substrate (1.2% w/v) is added to 6.0 mL NZY top agar at 50° C. and    mixed thoroughly. Keep solution at 50° C. until needed.-   4) Cell suspension is transferred to substrate/top agar solution and    gently mixed.-   5) Solution is immediately poured onto large (150 mm) NZY media    plate.-   6) Allow top agar to solidify completely (approximately 30 minutes),    then invert plate.-   7) Plate incubated at 39° C. for 8-12 hours.-   8) Plate observed for a clearing zone (halo) around a single plaque    (pure clone). If a single plaque cannot be isolated, adjust titer    and re-plate phage suspension.-   9) Single plaque with halo is cored out of agar and transferred to a    sterile micro tube. A large bore 200 μL pipette tip works well to    remove (core) the agar plug containing the desired plaque. To    amplify the titer, core 5 single active plaques into a micro tube.-   10) Phages are re-suspended in 500 μL SM buffer. 20 μL Chloroform is    added to inhibit any further cell growth.-   11) Pure phage suspension is incubated at room temperature for 4    hours or overnight before next step. The pure phage suspension is    stored at −80° C. by adding DMSO into the phage suspension (7% v/v).

Excision of Pure Clone

-   1) 100 μL of pure phage suspension is added to 200 μL E. coli MRF′    cells (OD600=1.0). To this, 1.0 μL of ExAssist helper phage (>1×106    pfu/mL; Stratagene) is added. Use 2059 Falcon tube for excision.-   2) Suspension is incubated at 37° C. for 15 minutes.-   3) 3.0 mL of 2× YT media is added to cell suspension.-   4) Incubate at 30° C. for at least 6 hours or overnight while    shaking.-   5) Tube transferred to 70° C. for 20 minutes. The phagemid    suspension can be stored at 4° C. for 1 to 2 months.-   6) 100 μL of phagemid suspension transferred to a micro tube    containing 200 μL of E. coli Exp 505 cells (OD600=1.0).-   7) Suspension incubated at 37° C. for 15 minutes.-   8) 300 μL of SOB is added to the suspension.-   9) Suspension is incubated at 37° C. for 30 to 45 minutes.-   10) 100 μL of suspension is transferred to a small (90 mm) LB media    plate containing Kanamycin (LB media with Kanamycin 50 μg/mL) for    Zap Express DNA libraries or Ampicillin (LB media with Kanamycin 100    μg/mL) for Zap II DNA libraries.-   11) The rest of suspension is transferred to another small LB media    plate.-   12) Use sterile glass beads to evenly distribute suspension on the    plate.-   13) Plates are incubated at 30° C. for 12 to 24 hours.-   14) Plate observed for colonies.-   15) Inoculate single colony into LB liquid media containing suitable    antibiotic and incubate at 30° C. for 12 to 24 hours.-   16) Glycerol stock can be prepared by adding 80% glycerol into    liquid culture (15% v/v) and stored at −80° C.

Activity Verification

-   1) 50 μL of liquid culture is transferred to a micro tube. Add 500    μL of 8% pH7 Amylopectin Azure into the same tube. Prepare 2 tubes    for each clone.-   2) Activity is tested at 50° C. for 3 hours and overnight. Use pH 7    buffer as control.-   3) Cool the test specimen at ice-water bath for 5 minutes.-   4) Add 750 μL of Ethaqnol and mixed thoroughly.-   5) Centrifuge at 13000 rpm (16000 g's) for 5 minutes.-   6) Measure OD of the supernatant at 595 nm.

RFLP Analysis

-   1) 1.0 mL of liquid culture is transferred to a sterile micro tube.-   2) Centrifuge at 13200 rpm (16000 g's) for 1 minute.-   3) Discard the supernatant. Add another 1.0 mL of liquid culture    into the same sterile micro tube.-   4) Centrifuge at 13200 rpm (16000 g's) for 1 minute.-   5) Discard the supernatant.-   6) Follow QIAprep spin mini kit protocol for plasmid isolation.-   7) Check DNA concentration using BioPhotometer.-   8) Use Sac I and Kpn I for first double digestion. Incubate at    37° C. for 1 hour.-   9) Use Pst I and Xho I for second double digestion. Incubate at    37° C. for 1 hour.-   10) Add Loading dye into the digested sample.-   11) Run the digested sample on a 1.0% agarose gel for 1-1.5 hours at    120 volts.-   12) View gel with gel imager. All clones with a different digest    pattern will be sent for sequence analysis.

Example 5: Assays for Amylases

The following example describes an exemplary assays for determining if apolypeptide is within the scope of the invention.

Preparation of Host Cultures

-   -   1. Start an overnight culture of XL1-Blue MRF′ host cells. Use a        single colony from a streak plate to inoculate 10 mL LB        supplemented with 20 ug/mL tetracycline. Grow overnight culture        shaking at 37° C. for at least 16 hours.    -   2. Using aseptic technique, inoculate a fresh 100 mL of LB_(Tet)        day culture with XL1-Blue MRF′ host from the overnight LB_(Tet)        culture.    -   3. Grow in a 37° C. shaker until the OD reaches 0.75-1.0.    -   4. Pellet host cells at 1000× g for 10 minutes and gently        resuspend in 10 mM MgSO₄ at OD5.    -   5. Dilute a small amount of host cells to OD1 for use in        titering and pintooling.    -   6. Host preparations can be used for up to 1 week when stored on        ice or at 4° C.        -   To shorten growth time for the day culture, use ½× the usual            Tet concentration in LB (½=10 ug/mL), or omit the antibiotic            altogether.        -   Do not use NZY when selecting with Tetracycline. The high            Mg⁺⁺ concentration in NZY medium renders Tet inactive.

Titering Lambda Libraries

-   -   7. Place three sterile microfuge tubes in a rack.    -   8. Aliquot 995 uL prepared host cells in one tube and 45 uL        prepared OD1 host cells into each of the two remaining tubes.    -   9. Add 5 uL of lambda library to the tube containing 995 uL host        cells and mix by vortexing. This results in a dilution factor of        200.    -   10. Prepare 1/2,000 and 1/20,000 dilutions by consecutively        adding 5 uL of previous dilution to the remaining two tubes        containing 45 uL prepared host cells. Mix by vortexing after        each dilution was made.    -   11. Allow phage to adsorb to host by incubating at 37° C. for 15        minutes.    -   12. Meanwhile, pipet 100 uL of prepared OD1 host cells to each        of three Falcon 2059 tubes.    -   13. Add 5 uL of each dilution to a separate 2059 tube containing        host cells.    -   14. Plate each by adding 3 mL top agar to each tube and quickly        pour over 90 mm NZY plates. Ensure a smooth, even distribution        before the top agar hardens.    -   15. Invert plates and incubate at 37° C. overnight.    -   16. Count plaques and calculate titer of the library stock (in        plaque forming units (pfu) per uL).

Lambda Microtiter Screening for Amylases

Preparation

-   -   1. Prepare a sufficient amount of XL1-Blue MRF′ host culture, as        described above, for the amount of screening planned. A culture        of 100 mL is usually sufficient for screening 2-3 libraries.    -   2. Autoclave several bottles compatible with the QFill2        dispenser. These are the wide-mouth Corning bottles, 250 mL        containing a sealing ring around the lip.    -   3. Make sure there are sufficient amounts of plates, top agar,        BODIPY starch, red starch solution, etc. available for the        screen.    -   4. Schedule the Day 2 robot run with a representative from        Automation.    -   Day 1    -   1. Label the 1536-well plates (black) with library screen and        plate number. Tough-Tags™ tube stickers, cut in half width-wise,        are ideal for labeling 1536 well plates.    -   2. Calculate volumes of library, host cells and NZY medium        necessary for the screen. This is easily done with an Excel        spreadsheet.    -   3. Combine the calculated volumes of lambda library and OD5 host        cells in a sterile 250 mL wide-mouth Corning bottle (containing        a sealing ring).    -   4. Allow adsorption to occur at 37° C. for 15 minutes.    -   5. Add the calculated volume of NZY medium and mix well. This is        referred to as the cell-phage-medium suspension.    -   6. Perform a concomitant titer by combining 50 uL of the        cell-phage-medium suspension with 250 uL of OD1 host cells in a        Falcon 2059 tube, then plating with 9 mL of top agar onto a 150        mm NZY plate. Incubate concomitant titer plate at 37° C.        overnight.    -   7. Load the dispenser with the remainder of the suspension and        array each labeled 1536-well plate at 4 uL per well. If the        dispenser leaves air bubbles in some wells, they can be removed        by centrifuging the plates at 200×g for 1 minute.    -   8. Add 0.5 uL of positive control phage to well position AD46 of        at least two of the assay plates. Use a strong amylase-positive        lambda clone for this purpose. The lambda versions of SEQ ID        NO.: 113 or SEQ ID NO.: 199 are good choices for positive        controls.    -   9. Incubate assay plates at 37° C. overnight in a humidified        (?95%) incubator.    -   Day 2    -   1. Count the pfu on the concomitant titer plate and determine        the average seed density per well (in pfu per well).    -   2. Pintool at least 2 plates of each library screen (preferably        the 2 containing positive controls) as follows:        -   a) Prepare 2 host lawn plates to act as a surface on which            to pintool: combine 250 uL of OD1 host cells with 2 mL 2%            red starch and plate with 9 mL top agar onto 150 mm NZY            plates. Hold each plate as level as possible as the top agar            solidifies in order to produce an even hue of red across the            plate.        -   b) Using a twice flame-sterilized 1536 position pintool,            replicate at least 2 of the screening plates onto the host            lawn plates.        -   c) Place the pintooled recipient plates in a laminar flow            hood with the lids off for about 15-30 minutes (to vent off            excess moisture).        -   d) Replace the lids and incubate inverted at 37° C.            overnight.    -   3. Prepare the 2× BODIPY starch substrate buffer as follows:        -   a) Calculate the total volume of 2× substrate buffer            solution needed for all screening plates at 4 uL per well            (including any extra deadspace volume required by the            dispenser) and measure this amount of 100 mM CAPS pH 10.4            into a vessel appropriate for the dispenser used.        -   b) Retrieve enough 0.5 mg tubes of BODIPY starch to produce            the required volume of 2× substrate buffer [calculated in            step a) above] at a final concentration of 20-30 ug/mL.        -   c) Dissolve each 0.5 mg tube in 50 uL DMSO at room            temperature, protected from light, with frequent vortexing.            This takes more than 15 minutes; some production lots of            BODIPY starch dissolve better than others.        -   d) Add 50 uL 100 mM CAPS buffer pH 10.4 to each tube and mix            by vortexing.        -   e) Pool the contents of all tubes and remove any undissolved            aggregates by centrifuging for 1 minute at maximum speed in            a microfuge.        -   f) Add the supernatant to the rest of the 100 mM CAPS buffer            measured in step a) above.        -   g) Protect the 2× substrate buffer from light by wrapping in            foil.    -   4. Take plates and substrate buffer to the automation room and        program the robot with the following parameters:        -   a) dispense 4 uL substrate buffer per well        -   b) 1^(st) read at 1 hour post-substrate, 2^(nd) read at 9            hours, and third read at 17 hours; with 37° C. incubation            between reads        -   c) excitation filter: 485 nm; emission filter: 535 nm        -   d) set the Spectrafluor gain at 70, or the optimal gain for            the batch of 2× substrate buffer prepared.        -   e) ensure that the incubator used will protect assay plates            from light.    -   Day 3    -   1. Check pintooled plates for clearings in the bacterial lawn at        all positions corresponding to wells on the associated assay        plate. Also check for clearings in the red starch in any of the        pin positions. If plates containing positive controls were used        for pintooling, you should be able to see a large clearing zone        in the red background. Be wary of contaminants that also form        clearing zones in red starch (see comment “Contaminants That        Form Clearing Zones in Red Starch” at end of Example).    -   2. Identify putative hits from the data file produced by the        robot computer. The KANAL program produced by Engineering        simplifies data analysis. As a rule of thumb, a putative hit is        characterized as a well having signal intensity rising at least        1.5 fold over background.    -   3. For each putative, remove 2 uL from the well and add to a        tube containing 500 uL SM buffer and 50 uL CHCl3. Vortex to mix        and store at 4° C. This solution will be referred to hereafter        as the 4e-3 stock. The original screening plates should be        stored at 4° C., protected from light, at least until breakouts        are complete.

This is a recommended method of breaking out putative hits. It is aliquid phase assay that relies on confirmation of activity on BODIPYstarch. Alternatively, putative hits can be plated directly onto solidphase plates containing red starch such that 2,000-3,000 pfu per hit areexamined for clearing zones. However, inability to observe clearingzones on red starch is not necessarily an indication that a putative hitwas a false positive. It would then need to be assayed using the formatin which it was originally identified (i.e., liquid phase using BODIPYstarch as substrate). In addition, very weak positives are more easilyidentified using the method detailed below.

-   -   Day 1    -   1. In a sterile 50 mL conical tube, combine 0.5 mL OD5 host        cells with 45.5 mL NZY. This will be referred to as the        host-medium suspension.    -   2. For each putative hit to be analyzed, aliquot 1 mL of        host-medium suspension into each of 3 three sterile microfuge        tubes.    -   3. Set the 12-channel pipetman in multidispense mode with an        aliquot size of 20 uL and an aliquot number of 2×. Mount the        pipetman with a clean set of sterile tips.    -   4. Pour about 1 mL of host-medium suspension into a new sterile        solution basin and load the multichannel pipetman.    -   5. Dispense 20 uL per well into the last row (row P) of a black        384-well plate (12 channels×2=24 wells). This row will be used        later for the controls.    -   6. Expel the remaining liquid in the tips by touching the tips        against the surface of the basin and pressing the RESET button        on the pipetman. Lay the pipetman down in a way to prevent        contamination of the tips. There is no need to change the tips        at this point.    -   7. Pour the remainder of the fluid in the basin into a waste        container (like a beaker) taking care to avoid splash-back        contamination.    -   8. For the first putative to be analyzed, take 111 uL of the        4e-3 stock (see Day 2 in Lambda Microtiter Screening for        Amylases) and add it to the first in a set of three tubes        containing 1 mL host-medium suspension (step 2). Vortex to mix.        This is Dilution A.    -   9. Take 111 uL of Dilution A and add to the next tube in the        set. Vortex to mix. This is Dilution B.    -   10. Take 111 uL of Dilution B and add to the last tube in the        set. Vortex to mix. This is Dilution C. You should now have        three dilutions of phage, where concentrations of each differ by        a factor of 10.    -   11. Pour the contents of Dilution C (the most dilute of the 3        samples) into the solution basin and load the multichannel        pipetman.    -   12. Dispense 20 uL per well into the first row of the 384-well        plate (12 channels×2=24 wells).    -   13. Expel the remaining liquid in the tips by touching the tips        against the surface of the basin and pressing the RESET button        on the pipetman. Lay the pipetman down in a way to prevent        contamination of the tips. There is no need to change the tips        at this point.    -   14. Empty the basin as described above.    -   15. Pour the contents of Dilution B into the same basin and load        the multichannel pipetman.    -   16. Dispense 20 uL per well into the second row of the 384-well        plate.    -   17. Perform steps 13-16 similarly to dispense Dilution A into        the third row of the plate.    -   18. After all three dilutions have been arrayed into the first 3        rows of the plate, discard all tips and the solution basin into        the biohazardous waste container.    -   19. Mount the pipetman with a clean set of sterile tips and open        a new sterile solution basin.    -   20. Repeat steps 8-19 for each remaining putative hit, using        remaining rows on the plate up to row O. Five putative hits can        be analyzed on one 384-well plate, with the last row (row P)        saved for the controls.    -   21. Add 0.5 uL of each control to a separate well. Use at least        2-3 separate controls, preferably covering a range of activity.    -   22. Incubate assay plates at 37° C. overnight in a humidified        (≧95%) incubator.    -   Day 2    -   1. Pintool all breakout plates onto a host lawn with red starch        using the same method described for Day 2 in Lambda Microtiter        Screening for Amylases, except that a 384 position pintool is        used.    -   2. Prepare the 2× BODIPY starch substrate buffer as follows:        -   a) Calculate the total volume of 2× substrate buffer            solution needed for all breakout plates at 20 uL per well            (including any extra deadspace volume required by the            dispenser) and measure this amount of 100 mM CAPS pH 10.4            into a vessel appropriate for the dispenser used.        -   b) Retrieve enough 0.5 mg tubes of BODIPY starch to produce            the required volume of 2× substrate buffer [calculated in            step a) above] at a final concentration of 20-30 ug/mL.        -   c) Dissolve each 0.5 mg tube in 50 uL DMSO at room            temperature, protected from light, with frequent vortexing.            This takes more than 15 minutes; some production lots of            BODIPY starch dissolve better than others.        -   d) Add 50 uL 100 mM CAPS buffer pH 10.4 to each tube and mix            by vortexing.        -   e) Pool the contents of all tubes and remove any undissolved            aggregates by centrifuging for 1 minute at maximum speed in            a microfuge.        -   f) Add the supernatant to the rest of the 100 mM CAPS buffer            measured in step a) above.        -   g) Protect the 2× substrate buffer from light by wrapping in            foil.    -   3. Dispense 20 uL per well into all breakout plates.    -   4. Wrap all plates in aluminum foil and incubate at room        temperature for 2-6 hours.    -   5. Read each plate in the Spectrafluor with the following        settings:        -   a) fluorescence read (excitation filter: 485 nm; emission            filter: 535 nm)        -   b) plate definition: 384 well black        -   c) read from the top        -   d) optimal gain        -   e) number of flashes: 3    -   6. On the resulting Excel spreadsheet, chart each putative's 3        rows in a separate graph and check for activity. Ensure that the        positives controls produced signals over background.    -   7. For each putative that appears to have a real signal among        the wells, harvest a sample from a positive well as follows:        -   a) Select a positive well from a row representing the            highest initial dilution.        -   b) Transfer 2 uL from that well into a tube containing 500            uL SM and 50 uL CHCl₃. This is referred to as the breakout            stock.        -   c) Store at 4° C.    -   8. Using methods previously described, plate about 10 uL of each        breakout stock onto 150 mm NZY plates using red starch. The        objective is to obtain several (at least 20) well-separated        plaques from which to core isolates.    -   Day 3    -   1. Check pintooled plates for an acceptable incidence of        clearings in the bacterial lawn corresponding to wells on the        associated assay plate. Also check for clearings in the red        starch in the positive controls and in any tested putatives. Be        wary of contaminants that also form clearing zones in red starch        (see below).    -   2. From the solid phase plates containing dilutions of breakout        stocks, core several isolated plaques, each into 500 uL SM with        50 uL CHCl₃. This is referred to as the isolate stock.    -   3. The isolate stocks can then be individually tested on BODIPY        starch using methods described above. This step can be skipped        if the plaque that was cored in step 2 produced a clearing zone        in the red starch background. The isolate stocks were then be        individually tested on BODIPY starch using methods described        above. However, this step may be skipped if the plaque that was        cored in step 2 produced a clearing zone in the red starch        background.    -   Excisions    -   Day 1    -   1. In a Falcon 2059 tube, mix 200 uL OD1 XL1-Blue MRF′ host, 100        uL lambda isolate stock and 1 uL ExAssist phage stock.    -   2. Incubate in 37° C. shaker for 15 minutes.    -   3. Add 3 mL NZY medium.    -   4. Incubate in 30° C. shaker overnight.    -   Day 2    -   1. Heat to excision tube to 70° C. for 20 minutes.    -   2. Centrifuge 1000×g for 10 minutes.    -   3. In a Falcon 2059 tube, combine 50 uL supernatant with 200 uL        EXP505 OD1 host.    -   4. Incubate in 37° C. shaker for 15 minutes.    -   5. Add 300 uL SOB medium.    -   6. Incubate in 37° C. shaker for 30-45 minutes.    -   7. Plate 50 uL on large LB_(Kan50) plate using sterile glass        beads. If the plates are “dry”, extra SOB medium can be added to        help disburse the cells.    -   8. Incubate plate at 30° C. for at least 24 hours.    -   9. Culture an isolate for sequencing and/or RFLP.

Growth at 30° C. reduces plasmid copy number and is used to mitigate theapparent toxicity of some amylase clones.

Contaminants that Form Clearing Zones in Red Starch

When using red starch on solid medium to assay phage for amylaseactivity, it is common to see contaminating colony forming units (cfu)that form clearing zones in the red starch. For pintooled plates, it isimportant to distinguish amylase-positive phage clones from thesecontaminants whenever they align with a particular well position. Thesource of the contaminating microbes is presumably the 2% red starchstock solution, which cannot be sterilized by autoclaving or byfiltering after preparation. It is thought that they are opportunisticorganisms that survive by metabolizing the red starch. In order toreduce these contaminants, use sterile technique when making 2% redstarch solutions and store the stocks either at 4° C. or on ice.

Screening of α-Amylases

Primary screening can be done using RBB-starch and/or FITC-starch assubstrates. Elevated active clones can be screened using RBB-starch assubstrate using induced cultures and by liquefaction assay. Stock andsequencing new elevated active clones based on liquefaction data can beperformed.

The amylases can be sorted into 96-well plates (or 384-well plates) at 1cell/well in 50 μl of LB+Tet. The plates can be incubated for 24 hoursat 30° C. Replicate plates can be made corresponding to each well forstorage. Forty-five (45)

of 12M urea is added to each well and the plates shaken for 10 minutes.Plates can be kept at room temp for at least 1 hour and the lysatestored at 4° C.

Assay Using RBB-Starch

75 μl of RBB-starch substrate (1% RBB-insoluble corn starch in 50 mMNaAc buffer, pH=4.5) can be added into each well of a new 96-well plate(V-bottom). Five micro-liters of enzyme lysate can be transferred intoeach well with substrate using Biomek or Zymark. The plates can besealed with aluminum sealing tape and shaken briefly on the shaker. Theplates can be incubated at 90° C. for 30 minutes, followed by cooling atroom temperature for about 5 to 10 minutes. One hundred micro-liters of100% ethanol can be added to each well, the plates sealed and shakenbriefly on the shaker. The plates can be then centrifuged 4000 rpm for20 minutes using bench-top centrifuge. 100 μl of the supernatant can betransferred into a new 96-well plate (flat bottom) by Biomek and readOD595.

Assay Using FITC-Starch

50 μl of substrate (0.01% FITC-starch in 100 mM NaAc buffer, pH=4.5) canbe added into each well of a new 384-well plate. 5 μl of enzyme lysatecan be transferred into each well with substrate and incubated the plateat room temperature overnight. The polarization change of the substrate,excitation 485 nm, emission 535 nm can be read for each well. 96 wellplates can be used for all assays.

Confirmation of New Active Clones

Each positive clone from screening can be grown and induced using astandard protocol. Each clone can be examined for growth (i.e., celldensity over time), activity at per cell level (RBB-starch assay andliquefaction assay), expression (protein gel) and solubility of protein(by microscope analysis). The confirmed new elevated clones can betransferred for fermentation. One of the clones have amylase activitygreater than that of the parent includes the enzyme set forth in SEQ IDNO:2 (encoded by SEQ ID NO:1).

Example 6: Characterization of Alpha Amylase pH Optimum and SpecificActivity Determination

The following example describes an exemplary method for determining if apolypeptide is within the scope of the invention, e.g., by alpha amylaseactivity pH optimum and specific activity determination.

The pH optimum for the hydrolysis of starch can be determined byreacting 50 uL of the GP-1, 0.35 U/ml, with a 100 ml of 1% solublestarch solution (0.0175 U/g of starch) for 30 minutes at 95° C. Thereducing ends generated in the liquefied starch solution can be measuredby the neocupronine assay, described herein. The percent hydrolysis ofcornstarch can be determined by measuring the number of sugar reducingends produced with the neocupronine assay. Seventy grams of buffersolution (pH 4-7) is weighed and 100 ppm of calcium added. Thirty gramsof cornstarch is mixed into the buffer solution to form a starch slurry.The enzyme can be added and the vessels sealed and incubated at 95° C.for 30 minutes with an initial heating rate of 6° C. per minute. A 1 mlsample can be extracted from the reaction beakers and analyzed by theneocupronine assay.

Example 7: Exemplary Protocol for Liquefying Starch and MeasuringResults

The following example describes exemplary protocols for liquefyingstarch using amylases of the invention.

Reaction Conditions: 100 mM PO₄ pH 6.5, 1% (w/w) liquefied starch DE 12at 55° C. Both TLC and HPLC assays were done to verify activity. Thedata from both assays showed that the clones were active.

pH profiles for the amylases can be run using phosphate buffer pHed from3.0-6.5, at 55° C. From the amount of observable hydrolysis, it can bevisually said that the clones were more active at certain pH values thanat other values at the above indicated reaction conditions:

An exemplary protocol for the saccharification of liquefied starch at pH6.5:

-   -   Adjust the pH of the liquefied starch to the pH at which the        saccharification(s) will be performed. Liquefy starch in 100 mM        sodium acetate buffer, pH 4.5 with 100 mM sodium phosphate salts        added so that before saccharification, the pH could be adjusted        to pH 6.5.    -   Weigh 5 gram samples of liquefied starch into tared bottles.    -   Use 0.04% (w/w) Optidex L-400 or approximately 400 mL of 1-10        diluted stock Optidex L-400 per 100 grams of liquefied starch.    -   Calculate the milligrams of Optidex L-400 contained in the 400        mL of 1-10 diluted stock Optidex L-400. Next, calculate the        volume of lysates needed to give the same concentration of        enzyme as the Optidex L-400.    -   Add enzymes to liquefied starch samples and incubate at desired        temperature (50 C.°). After 18 hours determine DE and prepare a        sample for HPLC analysis.

An exemplary DE Determination:

Exemplary Neocuproine Assay:

A 100 ml sample can be added to 2.0 ml of neocuproine solution A (40 g/Lsodium carbonate, 16 g/L glycine, 0.45 g/L copper sulfate). To this canbe added 2.0 ml of neocuproine solution B (1.2 g/L neocuproinehydrochloride-Sigma N-1626). The tubes can be mixed and heated in aboiling water bath for 12 minutes; cooled, diluted to 10 ml volume withDI water and the OD read at 450 nm on the spectrophotometer. The glucoseequivalent in the sample can be extrapolated from the response of a 0.2mg/ml glucose standard run simultaneously.

Exemplary HPLC Analysis:

Saccharification carbohydrate profiles are measured by HPLC (Bio-RadAminex HPX-87A column in silver form, 80° C.) using refractive indexdetection. Mobile phase is filtered Millipore water used at a flow rateof 0.7 ml/min. Saccharification samples are diluted 1-10 with acidifiedDI water (5 drops of 6 M HCl into 200 mL DI water) then filtered througha 0.45 mm syringe filter. Injection volume is 20 uL.

Exemplary TLC:

Reaction products can be w/d at hourly timepoints and spotted and driedon a TLC plate. Plates can be developed in 10:90 water:isopropanol andvisualized with either a vanillin stain or CAM stain and then heated toshow reducible sugars. The liquefied starch can be partially hydrolyzedto glucose in cases where activity is observed.

Example 8: Starch Liquefaction Using Amylases of the Invention

This example describes an exemplary method of the invention forliquefying starch using amylases of the invention.

Amylase concentrate can be prepared from fermentation broths by heattreatment, cell washing, alkaline extraction using microfiltration andultrafiltration (48% overall yield). The UF concentrate can beneutralized with acetic acid and formulated with 30% glycerol at pH 4.5.The activity level of the slurry formulation can be representative of acommercial product (120 U¹/g-0.5 kg/ton starch).

Standard Amylase Activity Assay

A 1 mL cuvette containing 950 μL of 50 mM MOPS pH 7.0 containing 5 mMpNP-α-D-hexa-(1→4)-glucopyranoside can be placed in the Peltiertemperature controller of the Beckman DU-7400 spectrophotometerpreheated to 80° C. The spectrophotometer is blanked at 405 nm and 50 μLof the enzyme solution is added to the cuvette, mixed well and theincrease in the OD_(405nm) was monitored over a one-minute interval. TheΔOD_(405nm/min) rate is converted to a standard unit of μmole/minutefrom the OD_(405nm) response of 50 μL of 1 μmole/mL PNP in 950 mL 50 mMMOPS at pH 7.0-80° C. One standard unit of thermostable alpha amylase(DTAA) is equal to the amount of enzyme that will catalyze the releaseof 1 μmole/mL/minute of pNP under the defined conditions of the assay.

Standard Glucoamylase Activity Assay

A 1 mL cuvette containing 950 μL of 50 mM MOPS pH 7.0 containing 5 mMpNP-α-D-glucopyranoside can be placed in the Peltier temperaturecontroller of the Beckman DU-7400 spectrophotometer preheated to 60° C.The spectrophotometer can be blanked at 405 nm and 50 μL of the enzymesolution is added to the cuvette, mixed well and the increase in theOD_(405nm) was monitored over a one-minute interval. The ΔOD_(405nm)/minrate is converted to a standard unit of μmole/minute from the OD_(405nm)response of 50 μL of 1 μmole/mL pNP in 950 mL 50 mM MOPS at pH 7.0-60°C. One standard unit of glucoamylase (DGA) is equal to the amount ofenzyme that will catalyze the release of 1 μmole/mL/minute of pNP underthe defined conditions of the assay.

Dextrose Equivalent Determination

The neocuproine method was used to measure the DE. Selected samples weremeasured by both the Invention procedure and by a GPC analyst using theGPC Fehlings procedure.

Neocuproine Assay

A 100 μl sample can be added to 2.0 ml of neocuproine solution A (40 g/Lsodium carbonate, 16 g/L glycine, 0.45 g/L copper sulfate). To this isadded 2.0 ml of neocuproine solution B (1.2 g/L neocuproinehydrochloride-Sigma N-1626). The tubes can be mixed and heated in aboiling water bath for 12 minutes; cooled, diluted to 10 ml volume withDI water and the OD read at 450 nm on the spectrophotometer. The glucoseequivalent in the sample can be extrapolated from the response of a 0.2mg/ml glucose standard run simultaneously.

The starch sample is diluted ˜1 to 16 with DI water with the exactdilution recorded. Ten milliliters of the diluted sample was added to 20mls of DI water. Ten milliliters of Fehlings solution A and B can beadded to the diluted starch. The sample can be boiled for 3 minutes andcooled on ice. Ten milliliters of 30% KI and 10 ml of 6N H₂SO₄ is added.The solution is titrated against 0.1N sodium thiosulfate. The titrantvolume is recorded and used to calculate the DE.

Residual Starch Determination

Post-saccharification samples can be checked for residual starch usingthe Staley iodine procedure.

Twenty grams of sample can be weighed into a large weigh dish. 45 μL ofIodine solution is added to the weigh dish and the starch solution ismixed well. Dark blue indicates the presence of starch, a lightblue-green indicates slight starch, light green indicates a trace ofstarch and yellow-red, absence of starch. Iodine solution is prepared bydissolving 21.25 grams of iodine and 40.0 grams of potassium iodide inone liter of water.

Oligosaccharide Profile

Liquefaction and saccharification carbohydrate profiles can be measuredby HPLC (Bio-Rad Aminex HPX-87C column in calcium form −80° C.) usingrefractive index detection.

Gel Permeation Chromatography

The molecular weight distribution can be determined by chromatography ona PL Aquagel-OH column with mass detection by refractive index (WatersModel 2410). A Viscotek Model T60 detector can be used for continuousviscosity and light scattering measurements.

Capillary Electrophoresis

Beckman Coulter P/ACE MDQ Glycoprotein System—separation of APTSderivatized oligosaccharides on a fused silica capillary—detection bylaser-induced fluorescence.

Primary Liquefaction

Line starch directly from the GPC process is pumped into a 60 liter feedtank where pH, DS (dry solids) and calcium level can be adjusted beforeliquefaction. The amylase is added to the slurry. The 32% DS slurry ispumped at 0.7 liter/minute by a positive displacement pump to the jet—apressurized mixing chamber where the starch slurry is instantaneouslyheated to greater than 100° C. by steam injection. The gelatinizedpartially liquefied starch is pumped through a network of piping (stillunder pressure) to give the desired dwell time (5 minutes) attemperature. The pressure is released into a flash tank and samples canbe taken. Samples are taken in duplicate.

Secondary Liquefaction

The liquefied starch can be collected in one liter glass bottles andheld in a water bath at 95° C. for 90 minutes.

Saccharification

Liquefied starch can be cooled to 60° C., the pH adjusted to 4.5 and thesamples treated with glucoamylase. Saccharification progress can bemonitored over time by HPLC.

Preparation of Liquefied Syrups for Analysis

These trials were run to obtain liquefied syrups at DE's of ˜12 and 18for three amylases; SEQ ID NO: 6, SEQ ID NO: 2, commercial B.licheniformis amylase. The syrups were saccharified with three levels ofglucoamylase. The liquefied syrups were also analyzed by HPLC and gelpermeation chromatography.

Saccharification

The liquefied syrups produced with each amylase can be adjusted toapproximately pH 2.5 with 6N HCl immediately after the 90 minutesecondary liquefaction to inactivate any residual amylase. The syrupscan be then adjusted to pH 4.5, placed in a 60° C. water bath andsaccharified with three levels of glucoamylase. The extent ofsaccharification can be monitored by HPLC at 18 to 88 hour time points.

The liquefied syrups can be saccharified with the standard dosage—0.04%of a double-strength glucoamylase—and two lower dosages (50% and 25%) tomonitor any differences in the saccharification progress.Saccharification Progress—% dextrose development vs time—0.04%glucoamylase.

The liquefied syrups were saccharified with the standard dosage—0.04% ofa double-strength glucoamylase—and two lower dosages (50% and 25%) tomonitor any differences in the saccharification progress. The glucoselevels are higher at earlier time points with the SEQ ID NO:2 syrups.Some of the difference is due to a higher starting point but themolecular weight profile difference is also a contributing factor (theoligosaccharides in the SEQ ID NO:2 liquefied syrups—being smaller andmore uniform—should be, and apparently are, better substrates forglucoamylase).

Saccharification Progress—% dextrose development vs time—0.04%glucoamylase

Amylase 18 hr 24 hr 40 hr 44 hr 88 hr Commercial 70.2 78.4 86.1 86.794.2 SEQ ID NO: 2 79 88.6 92.5 92.8 95.3 SEQ ID NO: 6 74.1 85.9 91.991.6 94.8

Saccharification Progress—% dextrose development vs time—0.02%glucoamylase

Amylase 18 hr 24 hr 40 hr 44 hr 88 hr B. licheniformis 54.5 66.7 76.177.2 90.9 Amylase SEQ ID NO: 2 60.1 72 84.8 85.3 93.6 SEQ ID NO: 6 57.170 84 86.5 92.5

Post-Saccharification Sugar Profile

In these studies and all previous saccharification studies, the finalglucose level achieved after saccharification of Invention amylase andB. licheniformis liquefied syrups is essentially identical. The DP2(maltose) level is also similar. These large fragments are poorsubstrates for glucoamylase and tend to be converted slowly, if at all,into smaller fragments and ultimately, glucose).

Glucose DP2 DP3 >DP7 SEQ ID NO: 2 95.25 2.39 1.13 0.91 Commercial 94.162.10 0.39 2.91 SEQ ID NO: 6 94.77 2.27 1.48 0.82

Molecular Weight Distribution

The molecular weight distribution of syrups liquefied to DE's of 12 and18 by the invention's amylase SEQ ID NO:2, and SEQ ID NO:6, andcommercial Bacillus licheniformis and commercial Bacillusstearothermophilus) were measured by gel permeation chromatography usingdetection by refractive index, light scattering and viscosity. Both thelicheniformis and stearothermophilus amylases generate a bimodaldistribution—the primary peak centered at 2000, a secondary peak at32,000 with a shoulder extending past the 160,000 range. The lowermolecular weight peak represents approximately 60% of the total mass ofthe sample. The invention amylases exhibit a single peak at 2000 withvery little above 30,000.

HPLC

The DE 12 and 18 syrups produced by the Invention and commercialamylases were analyzed by HPLC. Both techniques(chromatograms/electropherograms) produce fingerprints characteristic ofeach class of amylase; the oligosaccharide patterns are different forlicheniformis vs stearothermophilus vs the invention's amylases. Theinvention's liquefied syrups exhibit evidence of greater branching inthe oligosaccharides. HPLC only resolve the oligosaccharides in the<DP15 range—larger fragments are not visible in these techniques.Bacillus amylases are known to liquefy starch in a manner such that theamylopectin fraction is hydrolyzed less extensively than the amylosefraction. These >DP30 amylopectin fragments are contained in the highmolecular weight fraction centered at 32,000 and consequently, littleevidence of branching is seen in the HPLC analyses of the Bacillusliquefied syrups. The <DP15 oligosaccharides from Invention amylasescontain fragments from both amylose and amylopectin.

This example characterizes the liquefaction oligosaccharide patternsresulting from the invention's enzyme SEQ ID NO:2 and SEQ ID NO:6 versuscommercial Bacillus licheniformis and Bacillus stearothermophilusamylases. These results looked at the saccharification progress andfinal dextrose levels from syrups generated by invention and commercialamylases.

Three commercial enzymes, Genencor Spezyme AA, and two others allrequired more than double the recommended dosage to achieve the targetDextrose equivalent (DE). Dextrose equivalent (DE) is the industrystandard for measuring the concentration of total reducing sugars,calculated as D-glucose on a dry weight basis. Unhydrolyzed granularstarch has a DE of virtually zero, whereas the DE of D-glucose isdefined as 100.

This confirms the “double dosage” effect for all Bacillus amylases andgives more credence to the proposal that the observed dosage for SEQ IDNO:2 in the trials is also twice the value which would be required undermore normal conditions. The projected “normal” dosage, 60-70 Units/kilostarch at pH 4.5 to reach a 19 DE, is consistent with the laboratoryliquefaction data.

The oligosaccharide pattern generated by invention amylases is differentfrom the Bacillus profiles. The molecular weight distribution for theBacillus amylases (gel permeation chromatography with detection by lightscattering and viscosity) is bimodal with a substantial fraction at thevery high molecular weight range (>300,000) even at an 18DE. The SEQ IDNO:2 at 18DE exhibits a uniform distribution with nothing greater than20,000. This is consistent with the lower viscosity for Inventionsyrups. The DP (degrees of polymerization) profiles as measured by HPLCalso reflects this difference in action pattern.

In this study, as well as in the previous studies, the final glucoselevel after saccharification of Invention amylase liquefied syrups vsthe Bacillus syrups is the same for both cases. However, we have nowacquired sufficient saccharification data from internal as well as fromthe GPC studies to confirm that the non-dextrose residuals for theinvention amylases are different from the Bacillus amylase syrups.Although the dextrose and maltose levels are essentially the same forboth, the Invention amylases have a higher DP3 fraction but lower amountof the “highers” vs. the Bacillus enzyme. Consistent with the absence ofhigh molecular weight fragments after liquefaction, the postsaccharification Invention syrups have a lower content of the >DP7fraction.

Glucose DP2 DP3 >DP7 SEQ ID NO: 2 95.25 2.39 1.13 0.91 Commercial 94.162.10 0.39 2.91 SEQ ID NO: 6 94.77 2.27 1.48 0.82

SEQ ID NO:2 amylase concentrate was prepared from fermentation broths byheat treatment, cell washing, alkaline extraction using microfiltrationand ultraflitration (48% overall yield). The UF concentrate wasneutralized with acetic acid and formulated with 30% glycerol at pH 4.5.The activity level of the slurry formulation was representative of acommercial product (120 U¹/g-0.5 kg/ton starch).

Example 9: Starch Liquefaction at pH 4.5 Using Amylases of the Invention

The conversion of starch to glucose can be catalyzed by the sequenceaction of two enzymes: alpha-amylases of the invention to liquefy thestarch (e.g., the hydrolysis of high molecular weight glucose polymersto oligosaccharides consisting of 2 to 20 glycose units, typically adextrose equivalent of 10 to 12, by an amylase of the invention),followed by saccharification with a glycoamylase (which can be aglycoamylase of the invention). In one aspect, processing is in a cornwet milling plant producing a starch slurry having a pH or about 4.0 to4.5. In one aspect, the pH is raised, e.g., to 5.8 to 6.0 beforeliquefaction to accommodate an alpha amylase with a low pH activity andstability (which can be an alpha amylase of the invention). In oneaspect, amylases of the invention can liquefy starch at pH 4.5 todextrose equivalents ranging from 12 to 18; in one aspect, using alphaamylases of the invention at levels of about 3 to 6 grams per ton ofstarch. In this aspect, use of alpha amylases of the invention enablesstarch liquefaction to be conducted at pH 4.5.

In one aspect, starch liquefaction is conducted at pH 4.5 for 5 minutesat 105° C. to 90 minutes at 95° C. using amylases of the invention. Thequantity of enzyme was adjusted in order to adjust a target DE of 12 to15 after liquefaction. In one aspect, the liquefied starch is thensaccharified with a glucoamylase, e.g., an Aspergillis glucoamylase, forabout 48 hours at about pH 4.5 and 60° C. If the saccharified syrup didnot contain at least 95% glucose, the target liquefaction DE was raisedand the saccharification repeated until the liquefaction eventually didproduce a saccharified syrup containing more than 95% glucose. Theamylase protein required to produce a suitable liquefied feedstock forsaccharification was determined by PAGE.

Example 10: Amylase Ligation Reassembly

Nine fragments (each about 150 bp) were amplified from each of theparent clones SEQ ID NO: 6, SEQ ID NO: 66, SEQ ID NO: 67, covering thewhole open reading frame. The primers are provided in Table 1.

TABLE 1 SEQ ID NO: GAACACTAGTAGGAGGTAACTTATGGCAAAGTATTCCGAGCTCGAAG 11SpeI GAACGGTCTCATTCCGCCAGCCAGCAAGGGGATGAGCGG 12 BsaIGAACCGTCTCAAAACACGGCCCATGCCTACGGC 13 BsmBIGAACGTCTCACCTCGACTTCCACCCCAACGAGGTCAAG 14 BsmAIGAACGTCTCAGGCGCTTTGACTACGTGAAGGGC 15 BsmAIGAACGGTCTCAACAAGATGGATGAGGCCTTTG 16 BsaIGAACCGTCTCACGATATAATCTGGAACAAGTACCTTGC 17 BsmBIGAACCGTCTCAGAAGCACGAGCATAGTTTACTACG 18 BsmBIGAACCGTCTCAAAGGTGGGTTTATGTGCCG 19 BsmBIGAACGTCTCAGGAATCCAAATGGCGGATATTCCCGC 20 BsmAIGAACGGTCTCAGTTTATCATATTGATGAGCTCC 21 BsaIGAACCGTCTCAGAGGTAGTTGGCAGTATATTTG 22 BsmBIGAACGTCTCACGCCAGGCATCAACGCCGATG 23 BsmAI GAACGTCTCATTGTAGTAGAGCGGGAAGTC24 BsmAI GAACGGTCTCAATCGGTGTCGTGGTTTGCTAC 25 BsaIGAACCGTCTCACTTCCACCTGCGAGGTGGTC 26 BsmBI GAACCGTCTCACCTTCCAACCTTGCTCGAGC27 BsmBI TCGAGACTGACTCTCACCCAACACCGCAATAGC 28GAACACTAGTAGGAGGTAACTTATGGCCAAGTACCTGGAGCTCGAAGAGG 29 SpeIGAACGGTCTCATTCCCCCGGCGAGCAAGGGC 30 BsaI GAACCGTCTCAAAACACCGCCCACGCCTACGG31 BsmBI GAACGTCTCACCTCGACTTCCACCCCAAC 32 BsmAIGAACGTCTCAGGCGCTTCGACTACGTCAAGG 33 BsmAIGAACGGTCTCAACAAGATGGACGCGGCCTTTGAC 34 BsaIGAACCGTCTCACGATATAATTTGGAACAAGTACCC 35 BsmBIGAACCGTCTCAGAAGCACCGACATAGTCTAC 36 BsmBI GAACCGTCTCAAAGGTGGGTCTACGTTCCG37 BsmBI GAACGTCTCAGGAATCCATATTGCGGAGATTCCGGC 38 BsmAIGAACGGTCTCAGTTTATCATGTTCACGAGCTC 39 BsaIGAACCGTCTCAGAGGTAGTTGGCCGTGTACTTG 40 BsmBIGAACGTCTCAGCCATGCGTCAACGCCGATG 41 BsmAI GAACGTCTCATTGTAGTAGAGCGGGAAGTCG42 BsmAI GAACGGTCTCAATCGGTGTCGTGGTTTGCAACG 43 BsaIGAACCGTCTCACTTCCACCGGCGAGGTGGTCGTG 44 BsmBIGAACCGTCTCACCTTCCGGCCTTGCTCGAGCC 45 BsmBITCGAGACTGACTCTCAGCCCACCCCGCAGTAGCTC 46GAACACTAGTAGGAGGTAACTTATGGCCAAGTACTCCGAGCTGGAAGAGG 47 SpeIGAACGGTCTCATTCCTCCCGCGAGCAAGGG 48 BsaI GAACCGTCTCAAAACACCGCCCACGCCTATG49 BsmBI GAACGTCTCACCTCGACTTCCACCCGAACGAGC 50 BsmAIGAACGTCTCAGGCGCTTCGACTACGTCAAGG 51 BsmAIGAACGGTCTCAACAAGATGGACGAGGCCTTCG 52 BsaI GAACCGTCTCACGATATAATCTGGAACAAG53 BsmBI GAACCGTCTCAGAAGCACTGACATCGTTTACTACG 54 BsmBIGAACCGTCTCAAAGGTGGGTTTACGTTCCG 55 BsmBI GAACGTCTCAGGAATCCATATCGCCGAAAT56 BsmAI GAACGGTCTCAGTTTATCATGTTTATGAGC 57 BsaIGAACCGTCTCAGAGGTAGTTGGCCGTGTATTTAC 58 BsmBIGAACGTCTCACGCCAGGCATCGATGCCGAT 59 BsmAIGAACGTCTCATTGTAGTAGAGGGCGAAGTCAAAG 60 BsmAIGAACGGTCTCAATCGGTATCGTGGTTGGCTACAAAC 61 BsaIGAACCGTCTCACTTCCTCCGGCGAGGTTGTCATG 62 BsmBIGAACCGTCTCACCTTCCGGCTTTGCTTGAGGC 63 BsmBITCGAGACTGACTCTCACCCAACACCGCAGTAGCTCC 64CACACAGCAGCAACCAACCTCGAGACTGACTCTCASCC 65 BbvI

Conditions used for PCR were as follows: 3 min 94° C., (30 sec 94° C.;30 sec 55° C., 30 sec 68° C.)×30 cycles, followed by 10 min 68° C. PCRproducts corresponding to homologous regions from the three parents werepooled (1:1:1), cut with the appropriate restriction enzyme (see Table3), and gel-purified. Equal amounts of fragment pools were combined andligated (16° C.; overnight). The resulting 450 bp ligation products weregel purified and ligated to yield full length amylase genes. Theresulting full length products were gel-purified and PCR amplified usinga mixture of F1 primers SEQ ID NO.: 6, SEQ ID NO.: 66, SEQ ID NO.: 67and primer SEQ ID NO:65. Conditions used for PCR were as follows: 3 min94° C., (30 sec 94° C.; 30 sec 50° C., 60 sec 68° C.)×30 cycles,followed by 10 min 68° C. The resulting PCR products (˜1.4 kbp) werepurified, cut with SpeI and BbvI, gel-purified, ligated into pMYC(vector from Mycogen, cut with SpeI/XhoI), and transformed into E. coliTop10. Plasmid DNA from a pool of ˜21000 colonies was isolated andtransformed into Pseudomonas.

Screening of Reassembled α-Amylase

The transformed Pseudomonas fluorescens (MB214) containing pMYC derivedfrom the parent clones SEQ ID NO:6, SEQ ID NO:66, SEQ ID NO:68 weresorted to 96- or 384-well plates by FACS and treated with 6M urea.Primary screening using RBB-starch and/or FITC-starch as substrates wascarried out as described more fully below. Elevated active clones werescreened using RBB-starch as substrate using induced cultures and byliquefaction assay. Stock and sequencing new elevated active clonesbased on liquefaction data was performed.

The transformed reassembled amylase library (MB214 (Pf)), were collectedand sorted into 96-well plates (or 384-well plates) at 1 cell/well in 50μl of LB+Tet. The plates were incubated for 24 hours at 30° C. Replicateplates were made corresponding to each well for storage. Forty-five (45)μl of 12M urea was added to each well and the plates were shaken for 10minutes. Plates were kept at room temp for at least 1 hour and thelysate stored at 4° C.

Assay Using RBB-Starch

75 μl of RBB-starch substrate (1% RBB-insoluble corn starch in 50 mMNaAc buffer, pH=4.5) was added into each well of a new 96-well plate(V-bottom). Five micro-liters of enzyme lysate was transferred into eachwell with substrate using Biomek or Zymark. The plates were sealed withaluminum sealing tape and shaken briefly on the shaker. The plates wereincubated at 90° C. for 30 minutes, followed by cooling at roomtemperature for about 5 to 10 minutes. One hundred micro-liters of 100%ethanol was added to each well, the plates sealed and shaken briefly onthe shaker. The plates were then centrifuged 4000 rpm for 20 minutesusing bench-top centrifuge. 100 μl of the supernatant was transferredinto a new 96-well plate (flat bottom) by Biomek and read OD595.Controls: SEQ ID NO:6, SEQ ID NO:66, SEQ ID NO:68.

Assay Using FITC-Starch

Added 50 μl of substrate (0.01% FITC-starch in 100 mM NaAc buffer,pH=4.5) into each well of a new 384-well plate. Transferred 5 μl ofenzyme lysate into each well with substrate and incubated the plate atroom temperature overnight. The polarization change of the substrate,excitation 485 nm, emission 535 nm, was read for each well. Controls:SEQ ID NO: 6, SEQ ID NO: 66, SEQ ID NO: 68. 96 well plates can be usedfor all assays.

Confirmation of New Active Clones

Each positive clone from screening was grown and induced using astandard protocol. Each clone was examined for growth (i.e., celldensity over time), activity at per cell level (RBB-starch assay andliquefaction assay), expression (protein gel) and solubility of protein(by microscope analysis). The confirmed new elevated clones weretransferred for fermentation. One of the clones have amylase activitygreater than that of the parent includes the enzyme set forth in SEQ IDNO:2 (encoded by SEQ ID NO:1).

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

What is claimed is:
 1. A method of generating a variant amylasecomprising an amino acid sequence that is at least 90% identical to theamino acid sequence of SEQ ID NO:2 comprising: (a) providing parentclones comprising the nucleic acid sequence as set forth in SEQ ID NO:6,SEQ ID NO:66, and SEQ ID NO:68; (b) providing primers and amplifying theprimers to generate pools of fragments of parent clones; (c) combiningand ligating the pools of fragments of (b) to generate the variantamylase genes; (d) transform the variant amylase genes of (c) into ahost cell that expresses the variant amylase.
 2. The method of claim 1,wherein the primers comprise any one of the nucleic acid sequences of:SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15,SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20,SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25,SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30,SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35,SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40,SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45,SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50,SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55,SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60,SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, and SEQ IDNO:65.